Polymer conjugates of therapeutic peptides

ABSTRACT

The invention provides peptides that are chemically modified by covalent attachment of a water-soluble oligomer. A conjugate of the invention, when administered by any of a number of administration routes, exhibits characteristics that are different from the characteristics of the peptide not attached to the water-soluble oligomer.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application Ser. No. 61/153,966, filed 19Feb. 2009, to U.S. Provisional Patent Application Ser. No. 61/208,089,filed 18 Feb. 2009, to U.S. Provisional Patent Application Ser. No.61/192,672, filed 19 Sep. 2009, the disclosures of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

Among other things, the present invention relates to conjugatescomprising a therapeutic peptide moiety covalently attached to one ormore water-soluble polymers.

BACKGROUND OF THE INVENTION

In many ways, the chemical and biological properties of peptides makethem very attractive candidates for use as therapeutic agents. Peptidesare naturally occurring molecules made up of amino acid building blocks,and are involved in countless physiological processes. With 20 naturallyoccurring amino acids, and any number of non-naturally occurring aminoacids, a nearly endless variety of peptides may be generated.Additionally, peptides display a high degree of selectivity and potency,and may not suffer from potential adverse drug-drug interactions orother negative side effects. Moreover, recent advances in peptidesynthesis techniques have made the synthesis of peptides practical andeconomically viable. Thus peptides hold great promise as a highlydiverse, highly potent, and highly selective class of therapeuticmolecules with low toxicity.

A number of peptides have been identified as therapeutically promising;however in vitro results have often not proven to bear out in vivo.Significantly, peptides suffer from a short in vivo half life, sometimesmere minutes, making them generally impractical, in their native form,for therapeutic administration. Thus there exists a need in the art formodified therapeutic peptides having an enhanced half-life and/orreduced clearance as well as additional therapeutic advantages ascompared to the therapeutic peptides in their unmodified form.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides conjugates comprising atherapeutic peptide moiety covalently attached to one or morewater-soluble polymers. The water-soluble polymer may be stably bound tothe therapeutic peptide moiety, or it may be releasably attached to thetherapeutic peptide moiety.

The invention further provides methods of synthesizing such therapeuticpeptide polymer conjugates and compositions comprising such conjugates.The invention further provides methods of treating, preventing, orameliorating a disease, disorder or condition in a mammal comprisingadministering a therapeutically effective amount of a therapeuticpeptide polymer conjugate of the invention.

Additional embodiments of the present conjugates, compositions, methods,and the like will be apparent from the following description, examples,and claims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present invention. Additional aspects and advantagesof the present invention are set forth in the following description andclaims, particularly when considered in conjunction with theaccompanying examples and drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. KISS1.1: Cation exchange purification of the PEGylation reactionmixture.

FIG. KISS1.2: RP-HPLC analysis of purified[mono]-[mPEG-ButyrALD-30K]-[Kisspeptin-13].

FIG. KISS1.3 MALDI-TOF spectrum of purified[mono]-[mPEG-ButyrALD-30K]-[Kisspeptin-13].

FIG. KISS2.1. Typical reversed phase purification profile of[mono]-[mPEG-ButyAldehyde-10K]-[Kisspeptin-10].

FIG. KISS2.2 Purity analysis of mono-[ButyrAldehyde-10K]-[Kisspeptin-10]by Reversed Phase HPLC.

FIG. KISS2.3. MALDI-TOF spectrum of purifiedmono-[mPEG-butyraldehyde-10k]-[Kisspeptin-10].

FIG. KISS3.1. Typical reversed phase purification profile of[mono]-[mPEG-ButyAldehyde-30K]-[Kisspeptin-10].

FIG. KISS3.2. Purity analysis of mono-[ButyrAldehyde-30K]-[Kisspeptin-1]by Reversed Phase HPLC.

FIG. KISS3.3. MALDI-TOF spectrum of purifiedmono-[mPEG-Butyraldehyde-30K]-[Kisspeptin-10].

FIG. KISS4.1. Typical reversed phase purification profile ofmono-[mPEG2-CAC-FMOC-40K]-[Kisspeptin-10].

FIG. KISS4.2. Purity analysis of[mono]-[CAC-PEG2-FOMC-40K]-[Kisspeptin-10] by Reversed Phase HPLC.

FIG. KISS4.3. MALDI-TOF spectrum of purifiedmono-[CAC-PEG2-FMOC-40K]-[Kisspeptin-10].

FIG. KISS5.1. Typical reversed phase purification profile ofmono-[mPEG-SBC-30K]-[Kisspeptin-10].

FIG. KISS5.2. SDS-PAGE, with Coomassie blue staining) of purifiedmono-[mPEG-SBC-30K]-[Kisspeptin-10].

FIG. KISS5.3. Purity analysis of mono-[mPEG-SBC-30K]-[Kisspeptin-10] byReversed Phase HPLC.

FIG. KISS5.4. MALDI-TOF spectrum of purifiedmono-[mPEG-SBC-30k]-[Kisspeptin-10].

FIG. KISS6.1 Typical cation exchange purification profile ofmono-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54].

FIG. KISS6.2. Purity analysis of[mono]-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54] conjugate by ReversedPhase HPLC.

FIG. KISS6.3. SDS-PAGE with Coomassie staining of purified[mono]-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54].

FIG. KISS6.4. MALDI-TOF spectrum of purified[mono]-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54].

FIG. KISS8.1. Agonist activity at GPR54 for stable PEG conjugates ofKisspeptin 10, Kisspeptin 13, and Kisspeptin 54.

FIG. KISS8.2. Agonist activity at GPR54 for releasable PEG conjugate ofKisspeptin 10.

FIG. KISS8.3. Agonist activity at GPR54 for releasable PEG conjugate ofKisspeptin 10.

FIG. ZIC2.1: Cation exchange purification ofmono-mPEG-C2-FMOC-20K-ziconotide from the PEGylation reaction mixture.

FIG. ZIC2.2: RP-HPLC analysis of purifiedmono-mPEG-C2-FMOC-20K-ziconotide.

FIG. ZIC2.3: MALDI-TOF analysis of purifiedmono-mPEG-C2-FMOC-20K-ziconotide.

FIG. ZIC3.1: Cation exchange purification ofmono-mPEG-CAC-FMOC-40K-ziconotide from the PEGylation reaction mixture.

FIG. ZIC3.2: RP-HPLC analysis of purifiedmono-mPEG-CAC-FMOC-40K-ziconotide.

FIG. ZIC3.3: MALDI-TOF analysis of purifiedmono-mPEG-CAC-FMOC-40K-ziconotide.

FIG. ZIC4.1: Cation exchange purification ofmono-mPEG-SBA-30K-ziconotide from the PEGylation reaction mixture.

FIG. ZIC4.2: RP-HPLC analysis of purified mono-mPEG-SBA-30K-ziconotide.

FIG. ZIC4.3: MALDI-TOF analysis of purifiedmono-mPEG-SBA-30K-ziconotide.

FIG. ZIC5.1: Cation exchange FPLC chromatography of the PEGylationreaction mixture between ziconotide and mPEG-SBC-30K-NHS.

FIG. ZIC6.1. Mean (±SEM) percent specific binding of ziconotideconjugates to calcium channel, N-type, in rat cortical membranes.

FIG. BIP2.1: (SPA-2K)2-biphalin purification with CG-71S resin.

FIG. BIP2.2: RP-HPLC analysis of reconstituted (SPA-2K)2-biphalin.

FIG. BIP2.3. MALDI TOF MS analysis of reconstituted (SPA-2K)2-biphalin.

FIG. BIP3.1: (C2-20K)₂-biphalin purification with CG-71 S resin.

FIG. BIP3.2: RP-HPLC analysis of reconstituted (C2-20K)₂-biphalin.

FIG. BIP3.3 MALDI-TOF analysis of reconstituted (C2-20K)₂-biphalin.

FIG. BIP4.1: (CAC-20K)₂-biphalin purification with CG-71S resin.

FIG. BIP4.2: (CAC-20K)₂-biphalin re-purification with CG-71S resin.

FIG. BIP4.3: RP-HPLC analysis of reconstituted (CAC-20K)₂-biphalin.

BIP4.4: MALDI-TOF analysis of reconstituted (CAC-20K)₂-biphalin.

FIG. BIP5.1: RP-HPLC analysis of SBC-30K and biphalin conjugationreaction mixture.

FIG. BIP5.2. The purification of (SBC-30K)₂-biphalin from the reactionmixture.

FIG. BIP6.1. Competition binding assay of biphalin anddi-CAC-20K-biphalin conjugate at human (A) μ opioid and (B) δ opioidreceptors.

FIG. BIP6.2. Competition binding assay of biphalin anddi-C2-20K-biphalin, di-SBC-30K-biphalin, and di-SPA-2K-biphalinconjugate at human (A) μ opioid and (B) δ opioid receptors.

FIG. BNP2.1. PEGylation rate of BNP-32 with mPEG2-40 kDa Butyr-ALD.

FIG. BNP2.2. Typical purification profile for the 40 kDa mPEG2-Butyr-ALDmono-PEG conjugate of BNP-32.

FIG. BNP2.3. HPLC analysis of the 40 kDa mPEG2-Butyr-ALD mono-PEGconjugate of BNP-32.

FIG. BNP2.4. MALDI-TOF analysis of the 40 kDa mPEG2-Butyr-ALD mono-PEGconjugate of BNP-32.

FIG. BNP2.5. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofBNP-32 and purified [mono]-[mPEG2-Butyr-ALD-40K]-[BNP-32] conjugate.

FIG. BNP4.1. Typical cation-exchange purification profile of[mono]-[mPEG-Butyr-ALD-10K]-[BNP-32].

FIG. BNP4.2. SDS-PAGE analysis of BNP-32 and the purified[mono]-[mPEG2-Butyr-ALD-40K]-[BNP-32] conjugate.

FIG. BNP4.3. RP-HPLC analysis of the purified[mono]-[mPEG-Butyr-ALD-10K]-[BNP-32] conjugate.

FIG. BNP4.4. MALDI-TOF analysis of the purified[mono]-[mPEG-Butyr-ALD-10K]-[BNP-32] conjugate.

FIG. BNP5.1. Typical first cation-exchange purification profile for[mono]-[mPEG-SBC-30K]-[BNP-32].

FIG. BNP5.2. SDS-PAGE analysis of the purified[mono]-[mPEG-SBC-30K]-[BNP-32] conjugate.

FIG. BNP5.3. RP-HPLC analysis of the purified[mono]-[mPEG-SBC-30K]-[BNP-32] conjugate.

FIG. BNP5.4. MALDI-TOF analysis of the purified[mono]-[mPEG-SBC-30K]-[BNP-32] conjugate.

FIG. BNP6.1. Typical first cation-exchange purification profile of[mPEG2-C2-fmoc-NHS-40K].

FIG. BNP6.2. SDS-PAGE analysis of the purified[mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate.

FIG. BNP6.3. RP-HPLC analysis of the purified[mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate.

FIG. BNP6.4. MALDI-TOF analysis of the purified[mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate.

FIG. BNP7.1 shows the mean plasma concentration-time profiles of forC2-FMOC-PEG2-40K-BNP, its corresponding metabolite and released BNP.

FIG. BNP7.2 shows the non-released PEG-BNP levels after theadministration of the two non-releasable PEG constructs(ButyrALD-40K-BNP, ButyrALD-10K-BNP).

FIG. PRO2.1. Typical cation exchange purification profile ofmono-[mPEG2-CAC-FMOC-40K]-[PG-1].

FIG. PRO2.2. SDS-PAGE of purified[mono]-[CAC-PEG2-FOMC-NHS-40K]-[Protegrin-1].

FIG. PRO2.3. Purity analysis of [mono]-[CAC-PEG2-FOMC-40K]-[Protegrin-1]by RP-HPLC.

FIG. PRO2.4. MALDI-TOF spectrum of purifiedmono-[CAC-PEG2-FMOC-40K]-[Protegrin-1].

FIG. PRO3.1 Typical cation exchange purification profile ofmono-[mPEG-SBC-30K]-[PG-1].

FIG. PRO3.2. SDS-PAGE of purified [mono]-[mPEG-SBC-30K-]-[Protegrin-1].

FIG. PRO3.3. Purity analysis of [mono]-[mPEG-SBC-30K-]-[Protegrin-1] byRP-HPLC.

FIG. PRO3.4. MALDI-TOF spectrum of purified[mono]-[mPEG-SBC-30K-]-[Protegrin-1].

FIG. PRO4.1 Typical reversed phase purification profile of[Protegrin-1]-[PEG-di-ButyrAldehyde-5K]-[Protegrin-1].

FIG. PRO4.2. SDS-PAGE of purified[Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1].

FIG. PRO4.3. Purity analysis of[Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1] by reversed phaseHPLC.

FIG. PRO4.4. MALDI-TOF spectrum of[Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1].

FIG. PRO5.1. Typical cation-exchange chromatography profile ofdextran-butryaldehyde-40K-protegrin-1.

FIG. PRO5.2. SDS-PAGE analysis (4-12% gel) of purifieddextran-butryraldehyde-40K-protegrin-1.

FIG. PRO6.1: PG-1 and (ALD)₂2K conjugates purification with CM SepharoseFF resin.

FIG. PRO6.2: RP-HPLC analysis of (PG-1)-(ALD)₂2K-(PG-1).

FIG. PRO6.3: MALDI analysis of (PG-1)-(ALD)₂2K-(PG-1).

FIG. PRO7.1.1 and 7.1.2: ALD40K-PG-1 purification with SP Sepharose HPresin.

FIG. PRO7.2. SDS-PAGE of the purified and concentrated ALD40K-PG-1.

FIG. PRO7.3: RP-HPLC analysis of ALD40K-PG-1 (lot #YW-pgALD40K-01).

FIG. PRO7.4: MALDI analysis of ALD40K-PG-1 (lot #YW-pgALD40K-01).

FIG. PRO8.1: CG40K-PG-1 purification with SP Sepharose HP resin.

FIG. PRO8.2: RP-HPLC analysis of purified CG40K-PG-1.

FIG. PRO8.3: MALDI-TOF analysis of purified CG40K-PG-1.

FIG. PRO9.1. Hemolysis relative to the 100% hemolysis produced by 0.25%Triton X-100.

FIG. PRO9.1. Hemolysis by PEG reagent controls.

FIG. PRO9.3. Hemolysis at the maximum concentration.

FIG. PRO9.4. Hemolytic activities of PG-1

FIG. PRO10.1 and PRO10.2 show the mean plasma concentration-timeprofiles for CG-PEG₂-FMOC-40K-PG-1 and CAC-PEG₂-FMOC-40K-PG-1, theircorresponding PEG-metabolite and released Protegrin-1.

FIG. PRO10.3 shows the released Protegrin-1 levels after theadministration of the two releasable PEG constructs versus the level ofProtegrin-1 given as native protein at the same dose (mg/kg).

FIG. PRO10.4 shows the mean plasma concentration-time profiles formPEG₂-PG-1, PG-1[PEG_(2k)-PG-1, PG-1-PEG_(5k)-PG-1.

FIG. V2.1. Typical cation-exchange purification profile of[mPEG2-NHS-20K]-[V681(V13AD)].

FIG. V2.2. SDS-PAGE analysis of V681(V13AD) PEGylation.

FIG. V2.3. Purity analysis of [mono]-[mPEG2-NHS 20K]-[V681(V13AD)]conjugate by reverse phase HPLC.

FIG. V2.4. MALDI-TOF spectra for [mono]-[mPEG2-NHS 20K]-[V681(V13AD)].

FIG. V3.1. Typical cation-exchange purification profile of[mPEG-SMB-301(]-[V681(V13AD)].

FIG. V3.2. SDS-PAGE analysis of V681(V13AD) PEGylation and purificationon the SP ion-exchange column.

FIG. V3.3. Purity analysis of [mono]-[mPEG-SMB-30K]-[V681(V13AD)]conjugate by reverse phase HPLC.

FIG. V3.4. MALDI-TOF spectra for [mono]-[mPEG-SMB 30K]-[V681(V13AD)].

FIG. V4.1 shows the mean plasma concentration-time profiles for V681(V13AD), SMB-30K-V681 (V13AD), and NHS-20K-V681 (V13AD).

FIG. V5.1. Hemolysis relative to the 100% hemolysis produced by 0.25%Triton X-100.

FIG. C-PEP 2.1. Typical anion-exchange chromatography profile of[[mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)].

FIG. C-PEP 2.2. Purity analysis of[[mono]-[mPEG-ru-MAL-30K]C-peptide(S20C)] by reversed phase HPLC.

FIG. C-PEP2.3. MALDI-TOF spectrum for[mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)].

FIG. C-PEP3.1. Typical anion-exchange chromatography profile of[[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)].

FIG. C-PEP3.2. Purity analysis of[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)] by reversed phaseHPLC.

FIG. C-PEP3.3. MALDI-TOF spectrum for[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)].

FIG. C-PEP4.1. Typical anion-exchange chromatography profile of[mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)].

FIG. C-PEP4.2. Purity analysis of[[mono]-[C2-PEG2-FMOC-40K]C-peptide(S20C)] by reversed phase HPLC.

FIG. C-PEP4.3. MALDI-TOF spectrum for[mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)].

FIG. C-PEP5.1. Typical anion-exchange purification profile of[[mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20C)].

FIG. C-PEP5.2. Purity analysis of[mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20C)] by reversed phase HPLC.

FIG. C-PEP6.1 Typical anion-exchange chromatography profile ofdextran-butryaldehyde-40K-C-peptide(S20C).

FIG. C-PEP6.2. Concentration of fraction II from the anion-exchangechromatogram shown in FIG. c-pep6.1 by a second anion-exchangechromatography run.

FIG. C-PEP6.3. Purity analysis of[[mono]-[Dextran-40K]-[C-peptide(S20C)] by reversed phase HPLC.

FIG. C-PEP6.4. MALDI-TOF spectrum for[mono]-[Dextran-40K]-[C-peptide(S20C)].

FIG. OGF2.1. Typical CG71 S reversed phase purification profile ofmono-[mPEG2-CAC-FMOC-40K]-[OGF].

FIG. OGF2.2. Purity analysis of [mono]-[CAC-PEG2-FOMC-40K]-[OGF] byreversed phase HPLC.

FIG. OGF2.3. MALDI-TOF spectrum of purifiedmono-[mPEG2-FMOC-CAC-40K]-[OGF].

FIG. OGF3.1. Typical CG71S reverse phase purification profile ofmono-[mPEG2-C2-FMOC-40K]-[OGF].

FIG. OGF3.2. Purity analysis of mono-[mPEG2-FMOC-C2-40K]-[OGF] byreversed phase HPLC.

FIG. OGF3.3. MALDI-TOF spectrum of purifiedmono-[mPEG2-FMOC-C2-40K]-[OGF].

FIG. OGF4.1. Typical CG71S reversed phase purification profile ofmono-[mPEG-Butyraldehyde-30K]-[OGF].

FIG. OGF4.2. Purity analysis of mono-[mPEG-ButyrAldehyde-30K]-[OGF] byreversed phase HPLC.

FIG. OGF5.1. Typical CG71S reversed phase purification profile ofmono-[mPEG-epoxide-5K]-[OGF].

FIG. OGF5.2. Purity analysis of mono-[mPEG-epoxide-5K]-[OGF] by reversedphase HPLC.

FIG. OGF6.1. Typical CG71 S reversed phase purification profile ofmono-[mPEG-Butyraldehyde-10K]-[OGF].

FIG. OGF6.2. Purity analysis of mono-[mPEG-ButyrAldehyde-10K]-[OGF] byreversed phase HPLC.

FIG. OGF7.1. Competition binding assay of OGF at human (A) μ opioid and(B) δ opioid receptors: effects of incubation treatment conditions.

FIG. OGF7.2. Competition binding assay of OGF and PEG-OGF conjugates(released and unreleased) at human (A) μ opioid and (B) δ opioidreceptors.

FIG. OGF7.3. Competition binding assay of OGF and free PEGs at human (A)μ opioid and (B) δ opioid receptors.

FIG. INS1.1 Typical anion-exchange chromatography profile of theconjugation reaction mixture with partially acetylated insulin.

FIG. INS1.2 SDS-PAGE analysis of fractions containingdextran-butyrALD-40K-insulin collected from anion-exchangechromatography.

FIG. INS1.3 Concentration of purified dextran-butyrALD-40K-insulin byanion-exchange chromatography.

FIG. INS1.4. SDS-PAGE analysis of purified dextran-butyrALD-40K-insulin.

FIG. INS1.5 Typical anion-exchange chromatography profile of theconjugation reaction mixture with non-acetylated insulin.

In vitro binding of the Insulin-dextran conjugate.

FIG. INS3.1. Glucose levels after compound administration (0-8 hr).

DETAILED DESCRIPTION

As used in this specification and the intended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a polymer”includes a single polymer as well as two or more of the same ordifferent polymers; reference to “an optional excipient” or to “apharmaceutically acceptable excipient” refers to a single optionalexcipient as well as two or more of the same or different optionalexcipients, and the like.

In describing and claiming one or more embodiments of the presentinvention, the following terminology will be used in accordance with thedefinitions described below.

As used herein, the terms “therapeutic peptide” and “therapeuticpeptides” mean one or more peptides having demonstrated or potential usein treating, preventing, or ameliorating one or more diseases,disorders, or conditions in a subject in need thereof, as well asrelated peptides. These terms may be used to refer to therapeuticpeptides prior to conjugation to a water-soluble polymer as well asfollowing the conjugation. Therapeutic peptides include, but are notlimited to, those disclosed herein, including in Table 1. Therapeuticpeptides include peptides found to have use in treating, preventing, orameliorating one or more diseases, disorders, or conditions after thetime of filing of this application. Related peptides include fragmentsof therapeutic peptides, therapeutic peptide variants, and therapeuticpeptide derivatives that retain some or all of the therapeuticactivities of the therapeutic peptide. As will be known to one of skillin the art, as a general principle, modifications may be made topeptides that do not alter, or only partially abrogate, the propertiesand activities of those peptides. In some instances, modifications maybe made that result in an increase in therapeutic activities. Thus, inthe spirit of the invention, the terms “therapeutic peptide” or“therapeutic peptides” are meant to encompass modifications to thetherapeutic peptides defined and/or disclosed herein that do not alter,only partially abrogate, or increase the therapeutic activities of theparent peptide.

The term “therapeutic activity” as used herein refers to a demonstratedor potential biological activity whose effect is consistent with adesirable therapeutic outcome in humans, or to desired effects innon-human mammals or in other species or organisms. A given therapeuticpeptide may have one or more therapeutic activities, however the term“therapeutic activities” as used herein may refer to a singletherapeutic activity or multiple therapeutic activites. “Therapeuticactivity” includes the ability to induce a response in vitro, and may bemeasured in vivo or in vitro. For example, a desirable effect may beassayed in cell culture, or by clinical evaluation, EC₅₀ assays, IC₅₀assays, or dose response curves. In vitro or cell culture assays, forexample, are commonly available and known to one of skill in the art formany therapeutic peptides as defined and/or disclosed herein.Therapeutic activity includes treatment, which may be prophylactic orameliorative, or prevention of a disease, disorder, or condition.Treatment of a disease, disorder or condition can include improvement ofa disease, disorder or condition by any amount, including elimination ofa disease, disorder or condition.

As used herein, the terms “peptide,” “polypeptide,” and “protein,” referto polymers comprised of amino acid monomers linked by amide bonds.Peptides may include the standard 20 α-amino acids that are used inprotein synthesis by cells (i.e. natural amino acids), as well asnon-natural amino acids (non-natural amino acids may be found in nature,but not used in protein synthesis by cells, e.g., ornithine, citrulline,and sarcosine, or may be chemically synthesized), amino acid analogs,and peptidomimetics. Spatola, (1983) in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, Weinstein, ed., Marcel Dekker, NewYork, p. 267. The amino acids may be D- or L-optical isomers. Peptidesmay be formed by a condensation or coupling reaction between theα-carbon carboxyl group of one amino acid and the amino group of anotheramino acid. The terminal amino acid at one end of the chain (aminoterminal) therefore has a free amino group, while the terminal aminoacid at the other end of the chain (carboxy terminal) has a freecarboxyl group. Alternatively, the peptides may be non-linear, branchedpeptides or cyclic peptides. Moreover, the peptides may optionally bemodified or protected with a variety of functional groups or protectinggroups, including on the amino and/or carboxy terminus.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G.

The terms “therapeutic peptide fragment” or “fragments of therapeuticpeptides” refer to a polypeptide that comprises a truncation at theamino-terminus and/or a truncation at the carboxyl-terminus of atherapeutic peptide as defined herein. The terms “therapeutic peptidefragment” or “fragments of therapeutic peptides” also encompassesamino-terminal and/or carboxyl-terminal truncations of therapeuticpeptide variants and therapeutic peptide derivatives. Therapeuticpeptide fragments may be produced by synthetic techniques known in theart or may arise from in vivo protease activity on longer peptidesequences. It will be understood that therapeutic peptide fragmentsretain some or all of the therapeutic activities of the therapeuticpeptides.

As used herein, the terms “therapeutic peptide variants” or “variants oftherapeutic peptides” refer to therapeutic peptides having one or moreamino acid substitutions, including conservative substitutions andnon-conservative substitutions, amino acid deletions (either internaldeletions and/or C- and/or N-terminal truncations), amino acid additions(either internal additions and/or C- and/or N-terminal additions, e.g.,fusion peptides), or any combination thereof. Variants may be naturallyoccurring (e.g. homologs or orthologs), or non-natural in origin. Theterm “therapeutic peptide variants” may also be used to refer totherapeutic peptides incorporating one or more non-natural amino acids,amino acid analogs, and peptidomimetics. It will be understood that, inaccordance with the invention, therapeutic peptide fragments retain someor all of the therapeutic activities of the therapeutic peptides.

The terms “therapeutic peptide derivatives” or “derivatives oftherapeutic peptides” as used herein refer to therapeutic peptides,therapeutic peptide fragments, and therapeutic peptide variants thathave been chemically altered other than through covalent attachment of awater-soluble polymer. It will be understood that, in accordance withthe invention, therapeutic peptide derivatives retain some or all of thetherapeutic activities of the therapeutic peptides.

As used herein, the terms “amino terminus protecting group” or“N-terminal protecting group,” “carboxy terminus protecting group” or“C-terminal protecting group;” or “side chain protecting group” refer toany chemical moiety capable of addition to and optionally removal from afunctional group on a peptide (e.g., the N-terminus, the C-terminus, ora functional group associated with the side chain of an amino acidlocated within the peptide) to allow for chemical manipulation of thepeptide.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable and encompass any nonpeptidic water-solublepoly(ethylene oxide). Typically, PEGs for use in accordance with theinvention comprise the following structure “—(OCH₂CH₂)_(n)—” where (n)is 2 to 4000. As used herein, PEG also includes“—CH₂CH₂—O(CH₂CH₂O)_(n)—CH₂CH₂—” and “—(OCH₂CH₂)_(n)O—,” depending uponwhether or not the terminal oxygens have been displaced. Throughout thespecification and claims, it should be remembered that the term “PEG”includes structures having various terminal or “end capping” groups andso forth. The term “PEG” also means a polymer that contains a majority,that is to say, greater than 50%, of —OCH₂CH₂— repeating subunits. Withrespect to specific forms, the PEG can take any number of a variety ofmolecular weights, as well as structures or geometries such as“branched,” “linear,” “forked,” “multifunctional,” and the like, to bedescribed in greater detail below.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, more preferably aC₁₋₁₀ alkoxy group, and still more preferably a C₁₋₅ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. It must be remembered that the end-capping moiety may include oneor more atoms of the terminal monomer in the polymer [e.g., theend-capping moiety “methoxy” in CH₃—O—(CH₂CH₂O)_(n)— andCH₃(OCH₂CH₂)_(n)—]. In addition, saturated, unsaturated, substituted andunsubstituted forms of each of the foregoing are envisioned. Moreover,the end-capping group can also be a silane. The end-capping group canalso advantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) to which thepolymer is coupled can be determined by using a suitable detector. Suchlabels include, without limitation, fluorescers, chemiluminescers,moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions,radioactive moieties, gold particles, quantum dots, and the like.Suitable detectors include photometers, films, spectrometers, and thelike. The end-capping group can also advantageously comprise aphospholipid. When the polymer has an end-capping group comprising aphospholipid, unique properties are imparted to the polymer and theresulting conjugate. Exemplary phospholipids include, withoutlimitation, those selected from the class of phospholipids calledphosphatidylcholines. Specific phospholipids include, withoutlimitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

The term “targeting moiety” is used herein to refer to a molecularstructure that helps the conjugates of the invention to localize to atargeting area, e.g., help enter a cell, or bind a receptor. Preferably,the targeting moiety comprises of vitamin, antibody, antigen, receptor,DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell specificlectins, steroid or steroid derivative, RGD peptide, ligand for a cellsurface receptor, serum component, or combinatorial molecule directedagainst various intra- or extracellular receptors. The targeting moietymay also comprise a lipid or a phospholipid. Exemplary phospholipidsinclude, without limitation, phosphatidylcholines, phospatidylserine,phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine.These lipids may be in the form of micelles or liposomes and the like.The targeting moiety may further comprise a detectable label oralternately a detectable label may serve as a targeting moiety. When theconjugate has a targeting group comprising a detectable label, theamount and/or distribution/location of the polymer and/or the moiety(e.g., active agent) to which the polymer is coupled can be determinedby using a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, goldparticles, quantum dots, and the like.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may, however, containone or more monomers or segments of monomers that are naturallyoccurring, so long as the overall polymer structure is not found innature.

The term “water soluble” as in a “water-soluble polymer” is any polymerthat is soluble in water at room temperature. Typically, a water-solublepolymer will transmit at least about 75%, more preferably at least about95%, of light transmitted by the same solution after filtering. On aweight basis, a water-soluble polymer will preferably be at least about35% (by weight) soluble in water, more preferably at least about 50% (byweight) soluble in water, still more preferably about 70% (by weight)soluble in water, and still more preferably about 85% (by weight)soluble in water. It is most preferred, however, that the water-solublepolymer is about 95% (by weight) soluble in water or completely solublein water.

“Hydrophilic,” e.g, in reference to a “hydrophilic polymer,” refers to apolymer that is characterized by its solubility in and compatibilitywith water. In non-cross linked form, a hydrophilic polymer is able todissolve in, or be dispersed in water. Typically, a hydrophilic polymerpossesses a polymer backbone composed of carbon and hydrogen, andgenerally possesses a high percentage of oxygen in either the mainpolymer backbone or in pendent groups substituted along the polymerbackbone, thereby leading to its “water-loving” nature. Thewater-soluble polymers of the present invention are typicallyhydrophilic, e.g., non-naturally occurring hydrophilic.

Molecular weight in the context of a water-soluble polymer, such as PEG,can be expressed as either a number average molecular weight or a weightaverage molecular weight. Unless otherwise indicated, all references tomolecular weight herein refer to the weight average molecular weight.Both molecular weight determinations, number average and weight average,can be measured using gel permeation chromatography or other liquidchromatography techniques. Other methods for measuring molecular weightvalues can also be used, such as the use of end-group analysis or themeasurement of colligative properties (e.g., freezing-point depression,boiling-point elevation, and osmotic pressure) to determine numberaverage molecular weight, or the use of light scattering techniques,ultracentrifugation or viscometry to determine weight average molecularweight. The polymers of the invention are typically polydisperse (i.e.,number average molecular weight and weight average molecular weight ofthe polymers are not equal), possessing low polydispersity values ofpreferably less than about 1.2, more preferably less than about 1.15,still more preferably less than about 1.10, yet still more preferablyless than about 1.05, and most preferably less than about 1.03.

The term “active” or “activated” when used in conjunction with aparticular functional group refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

The terms “spacer moiety,” “linkage” and “linker” are used herein torefer to an atom or a collection of atoms optionally used to linkinterconnecting moieties such as a terminus of a polymer segment and atherapeutic peptide or an electrophile or nucleophile of a therapeuticpeptide. The spacer moiety may be hydrolytically stable or may include aphysiologically hydrolyzable or enzymatically degradable linkage. Unlessthe context clearly dictates otherwise, a spacer moiety optionallyexists between any two elements of a compound (e.g., the providedconjugates comprising a residue of a therapeutic peptide and awater-soluble polymer that can be attached directly or indirectlythrough a spacer moiety).

A “monomer” or “mono-conjugate,” in reference to a polymer conjugate ofa therapeutic peptide, refers to a therapeutic peptide having only onewater-soluble polymer molecule covalently attached thereto, whereas atherapeutic peptide “dimer” or “di-conjugate” is a polymer conjugate ofa therapeutic peptide having two water-soluble polymer moleculescovalently attached thereto, and so forth.

“Alkyl” refers to a hydrocarbon, typically ranging from about 1 to 15atoms in length. Such hydrocarbons are preferably but not necessarilysaturated and may be branched or straight chain, although typicallystraight chain is preferred. Exemplary alkyl groups include methyl,ethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms. “Cycloalkylene” refers to a cycloalkyl group that isinserted into an alkyl chain by bonding of the chain at any two carbonsin the cyclic ring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and soforth).

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl; C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. “Substituted awl” is aryl having oneor more noninterfering groups as a substituent. For substitutions on aphenyl ring, the substituents may be in any orientation (i.e., ortho,meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

An “organic radical” as used herein shall include alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,and substituted aryl.

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms, that may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucleophilic group” refers to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or with anelectrophile.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include but are not limited tocarboxylate ester, phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

“Releasably attached,” e.g., in reference to a therapeutic peptidereleasably attached to a water-soluble polymer, refers to a therapeuticpeptide that is covalently attached via a linker that includes adegradable linkage as disclosed herein, wherein upon degradation (e.g.,hydrolysis), the therapeutic peptide is released. The therapeuticpeptide thus released will typically correspond to the unmodified parentor native therapeutic peptide, or may be slightly altered, e.g.,possessing a short organic tag. Preferably, the unmodified parenttherapeutic peptide is released.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks. It must be pointed out that some linkages can behydrolytically stable or hydrolyzable, depending upon (for example)adjacent and neighboring atoms and ambient conditions. One of ordinaryskill in the art can determine whether a given linkage or bond ishydrolytically stable or hydrolyzable in a given context by, forexample, placing a linkage-containing molecule of interest underconditions of interest and testing for evidence of hydrolysis (e.g., thepresence and amount of two molecules resulting from the cleavage of asingle molecule). Other approaches known to those of ordinary skill inthe art for determining whether a given linkage or bond ishydrolytically stable or hydrolyzable can also be used.

The terms “pharmaceutically acceptable excipient” and “pharmaceuticallyacceptable carrier” refer to an excipient that may optionally beincluded in the compositions of the invention and that causes nosignificant adverse toxicological effects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a polymer-(therapeutic peptide) conjugatethat is needed to provide a desired level of the conjugate (orcorresponding unconjugated therapeutic peptide) in the bloodstream or inthe target tissue. The precise amount will depend upon numerous factors,e.g., the particular therapeutic peptide, the components and physicalcharacteristics of the therapeutic composition, intended patientpopulation, individual patient considerations, and the like, and canreadily be determined by one skilled in the art, based upon theinformation provided herein.

“Multi-functional” means a polymer having three or more functionalgroups contained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents of the invention willtypically contain from about 3-100 functional groups, or from 3-50functional groups, or from 3-25 functional groups, or from 3-15functional groups, or from 3 to 10 functional groups, or will contain 3,4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone. A“difunctional” polymer means a polymer having two functional groupscontained therein, either the same (i.e., homodifunctional) or different(i.e., heterodifunctional).

The terms “subject,” “individual,” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, rodents, simians, humans, farm animals,sport animals, and pets.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Substantially” (unless specifically defined for a particular contextelsewhere or the context clearly dictates otherwise) means nearlytotally or completely, for instance, satisfying one or more of thefollowing: greater than 50%, 51% or greater, 75% or greater, 80% orgreater, 90% or greater, and 95% or greater of the condition.

Unless the context clearly dictates otherwise, when the term “about”precedes a numerical value, the numerical value is understood to meanthe stated numerical value and also ±10% of the stated numerical value.

Turning now to one or more aspects of the invention, conjugates areprovided, the conjugates comprising a therapeutic peptide covalentlyattached (either directly or through a spacer moiety or linker) to awater-soluble polymer. The conjugates generally have the followingformula:

PEP-[-X-POLY]_(k)

wherein PEP is a therapeutic peptide as defined herein, X is a covalentbond or is a spacer moiety or linker, POLY is a water soluble polymer,and k in an integer ranging from 1-10, preferably 1-5, and morepreferably 1-3.

Therapeutic Peptides

As previously stated, the conjugates of the invention comprise atherapeutic peptide as disclosed and/or defined herein. Therapeuticpeptides include those currently known to have demonstrated or potentialuse in treating, preventing, or ameliorating one or more diseases,disorders, or conditions in a subject in need thereof as well as thosediscovered after the filing of this application. Therapeutic peptidesalso include related peptides.

In some embodiments of the invention, PEP is a therapeutic peptideselected from the group consisting of carperitide; alpha-neoendorphin;348U87; A-3847; A-4114; A-68552; A-75998; A-84861; AN-1792; AAMP-1;exenatide; AC-625; ACE-inhibitors, Aventis; ACE-inhibitors, SRI; ACTH,Amgen; ruprintrivir; AI-102; AI-202; NeuroVax; AI-402; AI-502; AIDStherapeutic vaccine, Repl; AIDS therapy, Inst Pasteur; AIDS vaccine,J&J; AIDS vaccine, Liposome Co; AIDS vaccine, Arana; AIDS vaccine,Peptimmune; AIDS vaccine, Sanofi Past-3; AIDS vaccine, Protherics; AIDSvaccine, SSVI; AIDS vaccine, SWFBR; AIDS vaccine, United-1; AIDSvaccine, United-2; AIDS vaccine-2, Yokohama; AIDS vaccine-3, NIH; AIDSvaccine-4, NIH; AIT-083; teduglutide; Skelite; Allotrap-2702;Alzheimer's imaging agent, Dia; AM-425; AN-238; AnergiX.RA; AnervaX.RA;AS-109; AV-9; AZM-134; addressin, Lilly; allergy vaccine, BioResearch;ambamustine; amylin antagonists, Amylin; anaritide analogues, Bio-Mega;anaritide, Bayer; anaritide, Bristol; anaritide, Aventis-2; anaritide,Astellas; anaritide, GlaxoSmithKline-2; anaritide, Aventis-1; anaritide,Mitsubishi Tanabe; anaritide, Novartis; anaritide, OmniGene; anaritide,Sankyo; anaritide, Scios; angiotensin II antagonists;anti-inflammatories, Affymax; anti-inflammatory peptide, BTG;anti-integrin peptides, Burnha; anti-TCR vaccines; antiallergy peptides,Ajin; antiallergy vaccine, Acambis-1; anticancer matrix, Telios;anticancer peptides, Micrologix; antiflammins; antifungal peptides, BTG;antifungal tripeptides, BTG; antiGnRH immunogen, Aphton; Gastrimmune;antirenin vaccine; antirheumatic peptides, Acambis; antithrombinpolypeptides; antiviral peptide, Bio-Mega; antiviral peptides,Non-indust; antiviral peptides, Yeda; apolipoprotein, NeuroSearch;apoptosis technology, Receptag; BCH-143; arthritis antigen; atrialnatriuretic peptide, Ph; atrial natriuretic peptide, Ra; avorelin;B-956; BCH-2687; BCH-2763; frakefamide; BIM-22015; BIM-26028; BIM-44002;BIO-1006; BIO-1211; Bio-Flow; BPC-15; Britistatin; BST-2001;bivalirudin; bombesin antagonist; brain natriuretic peptide; brainnatriuretic peptide, Phar; C-peptide analogues, UCB; C5a antagonist,Abbott; C68-22; Casocidin, Pharis; CBT-101; CCK(27-32), Organon; CD4,Genelabs; CD4-liposome conjugate, Sumito; CEE-04-420; CEP-079; CEP-903;CETP vaccine, Avant; mifamurtide; CGRP analogues, Asahi Chemical; CGRP,CSL; CGRP, Celltech; CGRP, Novartis; CGRP, Asahi Kasei; CGRP, SmithKlineBeecham; CGRP, Unigene; rusalatide acetate; CI-782; CKS-17; CMVpeptides, City of Hope; CNTF, Fidia; CP-95253; corticorelin acetate;CT-112, BTG; CT-1508; CTAP-III, Creative; CTP-37; PMD-2850; CVFM;CVT-857; CY-725; CY-726; CYC101; CYC103; CYC102; calcitonin, Peptitrol;calcitonin, Rockefeller; calciseptine; calcitonin analogues, SB;calcitonin, Amgen; calcitonin, Armour; calcitonin, Beaufour; calcitonin,Inhale; calcitonin, Bridgelock; calcitonin, microspheres; calcitonin,Nazdel; calcitonin, Novartis; calcitonin, nasal, Novartis; calcitonin,oral, Mannkind; calcitonin, Panoderm; calcitonin, Pharma Bissendorf;calcitonin, Pharmos; calcitonin, Anesiva; calcitonin, Aventis;calcitonin, Teijin; calcitonin, Teikoku; calcitonin, TheraTech;calcitonin, Yissum; calf thymus derived peptides; calpain inhibitors,ResCo; calphobindin I; cancer vaccines, Argonex; cargutocin;casokefamide; cekropin-P; chemokines, Dompe; tasidotin hydrochloride;ceruletide diethylamine; ceruletide, Fukuoka; cetrorelix acetate;chimaeric peptides, NIH; cholecystokinin, Ferring; collagenase IVinhibitors; collamers; contraceptive vaccine, Cephalo; contraceptivevaccine, Novarti; corplatin S compounds; corticoliberin, Pharma Bissend;corticoliberin, Salk; corticoliberin, Unigene; corticoliberin,Vanderbilt; D-21775; D-22213; Demegel; DAP inhibitors; DP-640; DP-107;DSIP; DU-728; Dynorphin A; daniplestim; defensins, LSB; desirudin;detirelix; dialytic oligopeptides; disagregin; E-2078; ECE inhibitor,SmithKline; ELS-1; EMD-73495; Enhancins; ecallantide; ES-1005; ES-305;echistatin; efegatran; eglin derivatives; elafin derivatives; elcatonin;eledoisin; encapsulated insulin, INSERM; endorphin, β-, Antigenics;endorphin, pancreatic; endorphin, β-, Mitsubishi; endorphin, β-, Amgen;endothelial cell growth factor; endothelin antagonists, ResCo;eptifibatide; examorelin; Factor VIII fragments, Pharma; FG-002; FG-003;FG-004; FG-005; FR-113680; FTS-Zn; fibrin-binding peptides, ISIS;fibronectin inhibitors, AstraZ; fibronectin-related peptide; follicularregulatory protein; G-4120; GAG-V3-VDP vaccine, Vern; GDL-peptides,Cytogen; EP-51216; GLP-1+exendin-4, NIH; GLP-1, Amylin; GLP-1,TheraTech; GM-1986; GM-CSF blocker, Hospira; GnRH-associated peptide;GPCR antagonists, NIH; GPIIb/IIIa antags, Selectide; GRF1-44; GRF,Lilly; GT2342; GT2501; GYKI-14451; galanin; gastrin antagonists;gastrin, Novo; glaspimod; glicentin; glucagon antagonists, Synvista;glucagon, Lilly; glucagon, ZymoGenetics; glucagon-121; glycoprotein 1balpha fragments; gonadorelin analogues, Syntex; gonadorelin antagonist,Ortho; gonadorelin preparations; gonadorelin, Arana; gonadorelin-MDPvaccine; goralatide; gp120-V3 peptides; growth factor peptides, Biothe;ENMD-0996; H-142, AstraZeneca; Her-2/Neu peptides, GSK; herpes simplexvaccine, Wistar; AIDS vaccine, Cel-Sci; HP-101; vitespen; HSV vaccine,Cel-Sci; HSV-1gD/vaccinia vaccine; heparin binding peptides, NCl;hepatitis-B receptor; hepatitis-B vaccine, Tokyo; hepatitis-B vaccine,Protherics; hepatitis-B vaccine-2, BTG; hirugen; I5B2; isegananhydrochloride; IgE peptides; IgG binding factor, Hoechst Ma;netarmftide; Insulin Aspart; Zorcell; icrocaptide; icatibant;immunomodulating peptides, Bio; infertility, E-TRANS; influenza vaccine,GSK-1; influenza vaccine, Yeda; instimulin; insulin analogue, Lilly;insulin analogues, Lilly; insulin analogues, Scios; insulin formulation,Pasteur; insulin glargine; insulin, Nektar, inhaled; insulin molecules,Novo; insulin oral, Inovax; insulin transdermal; insulin, Organon;insulin, ocular; insulin, AERx; insulin, AutoImmune; insulin, BEODAS;insulin, Biobras; insulin, Ferring Pharma; insulin, CJ Corp; insulin,Chiron; insulin, Chong Kun Dang; insulin, Sanofi Pasteur; insulin,Di-Arg, Hoechst Mario; insulin, E-TRANS; insulin, Forest; insulin,Hoechst, semisynth; insulin, Lilly, iodinated; insulin, Genentech,recombi; insulin, Provalis; insulin, Novartis; insulin, nasal; insulin,Ohio; insulin, Nazdel; insulin, Novo, synthetic; insulin, nasal, NovoNordisk; insulin, oral; insulin, buccal, Generex; insulin, Arana;insulin, Anesiva; insulin, Procter & Gamble; insulin, Qmax; insulin,Innovata; insulin, Roche; insulin, recombinant, Aventis; insulin,Shionogi; insulin, Shire; insulin, Spiros; insulin, SRI; insulin,Structured Biological; insulin, semisynthetic, Biobra; insulin,synthetic, Powerpatch; insulin, Zymo, recombinant; insulin,monocomponent, Novo; IL-1 receptor antagonist, Affym; interleukin-1B,Sclavo; interleukin-8 antags, Select; J015X; J018X; AG-1776; KNI-549;pralmorelin; KPI-022; katacalcin; ketomethylureas; L-346670; L-364210;L-659837; L-693549; L-709049; L-75; L-761191; L-histidyl peptides;LDV-containing peptides, Antiso; LEAPS-101; LHRH antagonists, Abbott;PD-6735; Lys-Phe; hLF1-11; lagatide; laminin A peptides, NIH; laminintechnology, NIH; lanreotide; leuprolide acetate, Atrigel; leuprorelin,Takeda; leuprorelin, Merck Serono; leuprorelin, DUROS; leuprorelin,Powerpatch; lipid-linked anchor technology; lysozyme metabolites, SPA;MCI-826; omiganan pentahydrochloride; MBP, ImmuLogic; MCI-536;MDL-104238; MDL-28050; Metascan; MMP inhibitors, NIH; MN-10006; MOL-376;MR-988; MSH derivatives; MUC-1 vaccine, Pittsburgh; malaria vaccine,Axis; malaria vaccine, Vernalis; malaria vaccine, Cel-Sci; malariavaccine, Roche; melanoma vaccine, Nobilon; meningitis vaccine, Acambis;mertiatide; metkephamide; metorphamide; monocyte chemotactic factor;montirelin hydrate; motyline; murabutide; muramyl dipeptide derivatives;myelopid; N-acetyl[Leu-28Leu-31]NPY24-36; N-carbobenzoxy peptides; NAGA;tiplimotide; opebecan; insulin detemir; liraglutide; Nona CCK; NP-06;NPC-18545; Nva-FMDP; nacartocin; natural peptide BPC, Pliva; nervegrowth factor, Synergen; nesiritide citrate; neuropeptides, Protherics;neuropeptides, Pfizer; neurotensin, Merck; neurotrophic factors,CereMedix; nifalatide; CL22, Innovata; nootropic, Yakult; nociceptin,Euroscreen; Org-2766; Org-30035; OSA peptides, Osteopharm; octreotide;opioid peptides, Unigene; osteogenic growth peptide; osteoporosispeptides, Telios; oxyntomodulin; P-113, Demegen; PACAP 27; PAPP;PD-83176; PD-122264; PD-132002; PEP-F; Penetratin; Peptigen agents;Phe-X-Gly, ResCo; PL-030; PN1 antagonists, Allelix; POL-443; POL-509;PPA, ResCo; PR-39; Prodaptin-M technology; PSP; tigapotide triflutate;PT-14; PT-5; semparatide; PTL-78968; parathyroid hormone fragments;pancreastatin; papillomavirus vaccine constru; parathyroid antagonist,Merck; enfuvirtide; peptide heterodimers, Cortech; peptide imaging,Diatide; pentapeptide 6A; pentigetide; peptide analogues, ResCo; peptide6, NY Medical College; peptide G, Arana; peptide inhibitors, ICRT;peptide T analogue, Carl; peptide T analogues; peptide T, Arana;peptide/drug vehicle, BTG; peptides, Sanofi-Aventis; peptides, Scios;peptides, Yeda; peptomers, NIH; pertussis vaccine-1, TRION; ph-914;ph-921; ph-9313; phospholipase inhibitors, Poli; prolactin, Genzyme;pramlintide; pranlukast; proinsulin, Lilly; proinsulin-2, Novartis;progenitor cell inhibitor, RCT; proinsulin fragments, Lilly; proinsulinanalogues, Lilly; proinsulin, Genentech; prostate cancer vaccine,United; prostate cancer vaccine, GSK; protirelin; protirelin, Takeda;Pseudomonas elastase inhibitor; QRS-10-001; QRS-5-005; Quilimmune-M;Retropep; RGD peptides; RHAMM targeting peptides, Cange; Ro-25-1553;RP-128; RSV vaccine, Avant; RSV vaccine, Acambis; RWJ-51438; TRH,Ferring; renin inhibitors, Pfizer-2; relaxin, Novartis; renininhibitors, INSERM; romurtide; rubella vaccine, Protherics; S-17162;S-2441; SC-40476; SC-44900; SDZ-CO-611; SIDR-1204; SK&F-101926;SK&F-110679; SLPI, Synergen; edotreotide; SP-1; SPAAT; SR-41476;SR-42128; SR-42654; SRIF-α; Streptococcus A vaccine, ID; Streptococcus Avaccine, SIGA; calcitonin, PPL; salmon calcitonin, Therapicon;sermorelin, Kabi; saralasin acetate; secretin, Eisai; secretin, Ferring;secretin, Wakunaga; sermorelin, Novartis; sermorelin peptides,Sanofi-Ave; sermorelin, Antigenics; sermorelin, Molecular Genetics;sermorelin acetate, Merck Ser; sermorelin, Sanofi-Aventis; sermorelin,Unigene; sinapultide; sleep inducing peptide, Bissen; small peptides,Centocor; somatoliberin, Takeda; PTR-3173; somatostatin analogue, Shira;somatostatin analogues, Merck; somatostatin analogues, Tulane;somatostatin derivatives; somatostatin, Merck Serono; somatostatin,Ferring; somatostatin, Arana; somatostatin, Sanofi-Synthelabo;somatostatin, BayerScheringPhar; T-205; Streptococcus A vaccine, Active;sulglicotide; syndyphalin; synthetic p16, Dundee; synthetic peptide BPC,Pliva; synthetic peptides, ICRT; T cell receptor peptide vaccin; T-118;T-786; T-cell receptor peptides, Xoma; T22; TA-3712; TASP inhibitors;TCMP-80; Tc-99m P215; Tc-99m P483H; Tc-99m P773; Tc-99m depreotide;Tc-99m-P280; TEI-1345; THF, Pfizer; Theradigm-HBV; Theradigm-HIV;Theratides; Stimuvax; ThGRF 1-29; tesarnorelin acetate; ThromboScan;TIMP, Creative BioMolecules; TIMP, Sanofi-Aventis; TJN-135; TNFinhibitor, Genelabs; TP-9201; TRH analogues, Roche; TRH, Daiichi; TRH,Japan Tobacco; TRH, Medicis; TRH, Arana; TRH-R, Medical Research Counci;TT-235; tabilautide; tendamistat; terlipressin; terlipressin, Nordic;teverelix; INKP-2001; thymic peptide; thymoleptic peptides; thymopentin;thymopentin analogues; thymosin alpha-2; thymosin 134; thymosin fraction5; tolerizing peptide, Acambis; trefoil peptides, ICRT; triletide;tuftsin, Abic; tuftsin, Sclavo; Type I diabetes vaccine, RCT; tyrosinekinase antags, ICRT; tyrosine-containing dipeptides; UA 1041; UA 1155;UA 1248; Uroguanylin, Pharis; urodilatin; V.F.; VIC, Astellas; VIPanalogues, TRION; VIP derivative, Eisai; VIP fusion protein, Kabi;vapreotide, immediate-release; varicella vaccine, ResCo; vitronectinreceptor antag; vicalcins; Mycoprex; YIGSR-Stealth; Yissum Project No.11607; Pharmaprojects No. 1088; Pharmaprojects No. 1113; PharmaprojectsNo. 1269; Pharmaprojects No. 1448; Pharmaprojects No. 1507;Pharmaprojects No. 1573; Pharmaprojects No. 1583; Pharmaprojects No.1626; Pharmaprojects No. 1779; Pharmaprojects No. 1797; PharmaprojectsNo. 1843; Pharmaprojects No. 1876; Pharmaprojects No. 1913;Pharmaprojects No. 1939; Pharmaprojects No. 1994; Pharmaprojects No.2043; Pharmaprojects No. 2044; Pharmaprojects No. 2063; PharmaprojectsNo. 2100; Pharmaprojects No. 2122; Pharmaprojects No. 2202;Pharmaprojects No. 2363; Pharmaprojects No. 2388; Pharmaprojects No.2425; Pharmaprojects No. 2476; Pharmaprojects No. 2527; PharmaprojectsNo. 2560; Pharmaprojects No. 2571; Pharmaprojects No. 2825;Pharmaprojects No. 2866; C-type natriuretic peptide, Sun; PharmaprojectsNo. 2909; Pharmaprojects No. 2912; Pharmaprojects No. 2913;Pharmaprojects No. 3009; Pharmaprojects No. 3020; Pharmaprojects No.3051; Pharmaprojects No. 3127; Pharmaprojects No. 3284; PharmaprojectsNo. 3341; Pharmaprojects No. 3392; Pharmaprojects No. 3393;Pharmaprojects No. 3400; Pharmaprojects No. 3415; Pharmaprojects No.3472; Pharmaprojects No. 3503; Pharmaprojects No. 3581; PharmaprojectsNo. 3597; Pharmaprojects No. 3654; Pharmaprojects No. 3667;Pharmaprojects No. 3777; Pharmaprojects No. 3862; Pharmaprojects No.3863; Pharmaprojects No. 3891; Pharmaprojects No. 3903; PharmaprojectsNo. 3939; Pharmaprojects No. 3963; Pharmaprojects No. 3989;Pharmaprojects No. 4004; Pharmaprojects No. 4093; Pharmaprojects No.4098; Pharmaprojects No. 4113; Pharmaprojects No. 4182; PharmaprojectsNo. 4209; Pharmaprojects No. 4246; Pharmaprojects No. 4251;Pharmaprojects No. 4300; Pharmaprojects No. 4323; Pharmaprojects No.4347; Pharmaprojects No. 4367; Pharmaprojects No. 4385; PharmaprojectsNo. 4402; Pharmaprojects No. 4445; Pharmaprojects No. 4544;Pharmaprojects No. 4625; Pharmaprojects No. 4626; Pharmaprojects No.4643; Pharmaprojects No. 4705; Pharmaprojects No. 4708; PharmaprojectsNo. 4766; GHRP-1, QLT; Pharmaprojects No. 4865; Pharmaprojects No. 491;Pharmaprojects No. 4915; Pharmaprojects No. 4936; Pharmaprojects No.494; Hematide; Pharmaprojects No. 4975; Pharmaprojects No. 5048;Pharmaprojects No. 5055; Pharmaprojects No. 5076; anti-HER2/neu mimetic,Cyclacel; Pharmaprojects No. 5131; Pharmaprojects No. 5173;Pharmaprojects No. 5181; Pharmaprojects No. 5200; Pharmaprojects No.5216; Pharmaprojects No. 5292; Pharmaprojects No. 5348; PharmaprojectsNo. 5356; Pharmaprojects No. 5412; DMP-444; Pharmaprojects No. 5657;Pharmaprojects No. 5728; Pharmaprojects No. 5839; Pharmaprojects No.5910; TGF-β antagonists, Inspiraplex; Pharmaprojects No. 5961;Pharmaprojects No. 5991; Pharmaprojects No. 6021; Pharmaprojects No.6063; Pharmaprojects No. 6083; PI-0824; RIP-3, Rigel; NBI-6024;Pharmaprojects No. 892; Pharmaprojects No. 955; IR-501; A6, Angstrom;leuprolide, ProMaxx; Orolip DP; edratide; 131-I-TM-601; ProsaptideTX14(A), Savient; insulin, Flamel; p1025; NIH; protein kinase R antags,NIH; GLP-1, Daiichi; EMD-249590; secretin, RepliGen; RANTES inhibitor,Milan; Pharmaprojects No. 6236; NY ESO-1/CAG-3 antigen, NIH; BILN-504SE; NIPs, RCT; insulin, Biphasix; ZRXL peptides, Novartis; BIM-23190;leuprorelin, TheriForm; β-amyloid peptides, CeNeS; oglufanide disodium;amyloid inhibiting peptides, Ax; iprP13; PN-277; differentiationinducers, Topo; immune privilege factor, Proneu; TASP-V; anticancervaccine, NIH; Pharmaprojects No. 6281; HAV peptide matrix, Adherex;calcitonin, oral, Biocon; analgesic, Nobex; PTH 1-34, Biocon; insulin,oral, Biocon-2; BLS-0597; leuprorelin, Depocore; IDPS; AIDS vaccine,Hollis-Eden; insulin, NovaDel; insulin, Orasome; Pharmaprojects No.6310; TRP-2. NIH; Pharmaprojects No. 6320; Re-188 P2045; calcitonin,Inovio; golotimod; angiotensin-II, topical, Trine; ETRx-101; antiallergyvaccine, Acambis-2; Tc-99m-P424; Tc-99m-P1666; insulin, Transfersome;Yissum Project No. 11649; SP(V5.2)C; melanoma vaccine, Therion-2;insulin Aspart, biphasic, Novo; Tat peptide analogues, NIH;Pharmaprojects No. 6365; Pharmaprojects No. 6373; Ramot project No. 981;ESP-24218; Pharmaprojects No. 6395; calcitonin, oral, Emisphere;omiganan, topical; AIDS vaccine, United-3; leuprorelin, Archimedes;HPV16 E6+E7 vaccine, NIH; peptide vaccine, NCl; Chlamydia vaccines,Argonex; delmitide acetate; RSV vaccine, Pierre Fabre-2; F-50040;CPI-1500; AIDS vaccine, BioQuest; insulin, BioSante, inhaled;antiangiogenics, GPC; TNF degradation product, Oncot; insulin,Emisphere; ozarelix; bremelanotide; Pseudomonas vaccine, Millenium; AIDSvaccine, CIBG; AIDS vaccine, Wyeth Vaccines-3; HCV serine proteaseinhib, BI; insulin, Wockhardt; cat PAD, Circassia; NOV-002; PPI-3088;insulin 24 hr, Altea; AP-811; hNLP, Pharis; ANUP-1, Pharis; serineprotease inhibs, Pharis; Pharmaprojects No. 6523; respiratory mucusinhibitor, Em; CLX-0100; AIDS vaccine, Panacos; SPHERE peptide vaccine,Genzyme; P-16 peptide, Transition; EP-51389; insulin, ProMaxx; ET-642;P-50 peptide, Transition Ther; Famoxin; insulin, Alkermes, inhaled; GPCRpeptide ligand, Synaptic; DiaPep227; alpha-1-antitrypsin, Cortech;IC-41; tuberculosis vaccine, Intercell; immunosuppressant vaccine, Aixl;malaria vaccine, NYU-2; netupitant; AG-702; insulin, AeroDose;anti-inflammatory, TapImmune; insulin glulisine; GPG-NH₂; hepatitis-Btherapy, Tripep; Staphylococcus therapy, Tripep; angiogenesis inhibitor,Tripep; bone marrow inhibitor, Tripep; melanoma vaccine, Biovector;lipopeptides, Cubist; ABT-510; parathyroid analogue, Unigene; Adageon-E;A-443654; CJC-1131; FE200 665; insulin, TranXenoGen; Gilatide; TFPI,EntreMed; desmopressin, Unigene; leuprorelin, oral, Unigene;antimicrobials, Isogenica; insulin, oral, Unigene; metastin; TRI-1144;DBI-4022; HM-9239; insulin, Bentley, intranasal; F-992; ZP-10; E1-INT;DEBIO-0513; spinal cord injury vacc, Weizrn; DAC:GLP-2; uPAR inhibitors,Message; MBP-8298; PL-14736; anaritide peptides, BTG; SP-1000,Samaritan; leuprorelin, Ardana; melanocyte modulators, IsoTis; HF-1020;leucocyte immobilizing peptide; Dentonin; MET-1000; SGS-111; 5-Helix;HPV vaccine, Ludwig; caries vaccine, Forsyth; taltobulin; ATN-161; T05;LY-307161; S pneumoniae vaccine, Milleniu; Alphastatin; anticancerpeptides, Wockhardt; PGN-0052; INNO-201; leuprolide, Nektar; insulin,BioSante, oral; ADD-9903; viral vaccines, Bio-Virus; AOD-9604;calcitonin, oral, Pfizer; insulin, INJEX; ETD-XXXX; analgesic, Sigyn;anti-infectives, AM-Pharma; human AMPs, AM-Pharma; INGAP peptide;osteomyelitis peptides, AM-Phar; XOMA-629; XMP-293 derivatives;BlockAide/VP; EradicAide; BlockAide/CR; VAC-12; leuprolide, oral, DORBioPharm; synthetic erythropoiesis pro; β-amyloid vaccine, Intellect;CEL-1000; sincalide; PankoPep; albiglutide; insulin, Bharat;leuprorelin, Norwood; Reversin 121, Solvo; SB-144; SB-29, STiL; cancervaccine, Sedac; SDT-021; malaria vaccine, Sedac, ther; malaria vaccine,Seda, prophyl; hepatitis-C cellular ther, Seda; Factor XIIIa inhib,Curacyte; insulin, Micronix; AIDS vaccine, Antigen Express-1; exenatideLAR; AIDS vaccine, Bionor Immuno-1; GV-1002; GV-1001; MSI vaccine,GemVax; PEP-14; PV-267; antibacterials, Provid; hepatitis-B vaccine,Innovata; BA-058; BIM-51077; malaria vaccine, Immunogenics; TM-701;VG-104; AC-162352; antivirals, Genencor; leuprolide acetate, Voyager;calcitonin, nasal, Archimedes; insulin, nasal, West; calcitonin, oral,Unigene; calcitonin, nasal, Unigene; IMX-735; IMX-775; PPI-01; anti-IgEpeptide, Allergy Ther; BZK-111; TH-0318; Enkastim; antibiotics, Bayer;Cerebrolysin; colorectal cancer therapy, IDM; wound growth factor,NephRx; JPD-105; osteoporosis drugs, Ferring; PN-951; CZEN-002; ZP-120;pasireotide; HerVac; CTT; LLG peptide, CTT; Pharmaprojects No. 6779;meptides, Senexis; Q-8008; FX-06; PhG-alpha-1; insulin, oral, Biocon;PP-0102; GTP-010; PAR-2 antagonists, EntreMed; parathyroid analogue,Zelos; K-1020; CTCE-9908; CTCE-0214; urocortin-II, Neurocrine;telomerase vaccine, Dendreon; AKL-0707; PYY3-36, Nastech; prostatecancer vaccine, Pepsca; AEZS-130; LYN-001; CUV-1647; AL-108; AL-309;HNTP-15; BIM-28131; CSF-G agonists, Affymax; IL-5 antagonists, Affymax;TRAIL agonists, Affymax; IgE inhibitors, Affymax; TM-801; TM-901;BN-054; APTA-01; HB-107; AVE cancer vaccine; PxSR; STD peptides, Helix;CF anti-infectives, Helix; HB-50; Homspera; S-0373; PYY3-36, oral,Emisphere; XG-101; XG-201CS; XG-102; insulin, oral, Coremed; Alzheimer'svaccine, Prana; AIDS vaccine, Bionor Immuno; leuprolide acetate,ALZAmer; AUX-202; AR-H044178; PYY3-36, Thiakis; lanreotide SR; malariavaccine, Pevion; Alzheimer's vaccine, Pevion; melanoma vaccine, AntigenExpr; melanoma vaccine, Pevion; OGP-(10-14)-L; ABS-13; ABS-17; cancertherapeutics, Argolyn; substance P-saporin; diabetes therapeutic, Thera;CGX-1051; OTS-102; Xen-2174; insulin, inhaled, Coremed; WP9QY;osteoporosis treatment, Fulcr; AHNP, Fulcrum; insulin, Technosphere,Mannkind; FX-07; CBP-501; E7 vaccine, Neovacs; LSI-518P; aviptadil,Mondobiotech; anticancer peptide, OrthoLogic; AL-209; OP-145; AT-001;AT-008; CHP-105; AMEP, BioAlliance; cardiovascular ther, Argolyn;TEIPP-03; mental retardation ther, Argol; IMX-002; IMX-942; NLC-001;octreotide, Indevus; DRF-7295; opioid peptide derivatives, Ka; CDX-110;ALT-212; desmopressin, Orexo; IMA-901; obinepitide; TM-30335; HIVtherapy, OyaGen-1; calcitonin, oral, ThioMatrix; insulin, oral,ThioMatrix; BRx-00585; Insulin Aspart, biphasic-2, No; CG-55069-11;GLP-1, Emisphere; linaclotide acetate; NPT-002; terlipressin, OrphanTherapeut; ZT-153; SciClone; FGLL; Syn-1002; MIP-160; PI-2301; PI-3101;BDM-E; insulin, Medtronic; ST-03; TH-0312; hepatitis-C vaccines, Kochi;cetrorelix acetate, once-weekly; RPI-MN; neurodegenerative ther,Recepto; RPI-78M; β-amyloid inhibitor, Alzhyme; DMI-3798; DMI-4983;ruzam; CT-319; EN-122004; glyponectin; EN-122001; EN-122002; KAI-9803;insulin, Advancell; larazotide acetate; calcitonin, oral, Bone Medical;parathyroid hormone, Bone Medi; calcitonin, Merrion; desmopressin,Merrion; acyline, Merrion; IMX-503; AP-214; Streptococcus vaccine,Vaccine; cytomegalovirus vaccine, Vacc; RHS-08; AG-707; antiallergics,Phylogica; PYC-36S; anticancers, Phylogica; Glypromate; NNZ-4945;calcitonin, intranasal, ITI; Peptide T, Advanced Immuni T; APTA-O₂;CGRP, Akela; TKS-1225; GalR2 peptide agonist, NeuroTa; botulinumvaccine, Emergent; HIV fusion inhibitors, Sequoia; AL-208; APP-018;BKT-RP3; smallpox vaccine, Antigen Expr; CMLVAX-100; INNO-105; insulin,Intravail; leptin, Intravail; calcitonin, Intravail; somatropin,Intravail; heparin, Intravail; erythropoietin, Intravail; CT-201;telomerase variants, GemVax; INT, transplantation; INT-3; SPI-1620;BIO-037; anticancers, Bracco; BIO-023; ZT-100; MC-4R agonists, Lilly;LT-1951; PTH (1-34), IGI; CGRP, VasoGenix; BIO-145; BIO-142; stem cellfactor, Affymax; VEGFR-2 antagonist, Affymax; KGF receptor agonist,Affymax; YM-216391; AT-007; AT-011; EK2700; EK900-1800; EK900-12; FGLm;ABS-201; Mdbt-12; autoimmune therapy, Antigen; VX-001; IPP-102199;IPP-201101; CTA1-DD; Factor VIIa inhibitor, ProTher; antiangiogenic,ProTherapeutic; IMT-1012; colon cancer vaccine, Immunoto; prohanin,ProTherapeutics; smallpox vaccine, BioDefense; heart failure therapy,ElaCor; PA-401; 802-2; insulin, nasal, Nastech; SEN-304; IMA-920;IMA-940; IMA-910; influenza vaccine, Antigen, H₅N₁; Primacoll;octreotide, PR Pharmaceuticals; female infertility th, Vyteris; FAR-404;athlete's foot therapy, Helix; leishmaniasis ther, Helix; INNO-305;ALS-O₂; sNN-0465; N,N-5401; TRI-999; Org-214444; Org-33409; IMA-930;YH-APC; PYC-35B; Rev-D4F; insulin, Phosphagenics; coeliac disease ther,Nexpep; coeliac disease therapy, BTG; exendin-4, PC-DAC; exenatide,nasal spray; CAP-232; ACE-011; Cardeva; BL-3020; FM-TP-2000; GGTI-2418;TM-30339; DP-74; DP-68; PPH ther, GeoPharma; MPL-TLB100; AZX-100;Alloferon; S2; S3; S4; PAC-G31P; PAC-745; PAC-525; PAC-113; VEBv;lipopeptide, Combinature; mondopeptide-1; mondopeptide-2;mondopeptide-2+mondopeptide-3; mondopeptide-4; MLIF; carfilzomib;Affitope AD-01; LT-ZP001; LT-ZMP001; CGX-1204; C3d, Enkam; C5aantagonist, Eucodis; adenocarcinoma vacc, ImmvaRx; insulin, oral,Apollo; renin inhibitors, Servier; Factor VIIa, GTC; ABS-212; NAFB001;NAFB002; insulin, MediVas; ZT-181; anti-inflammatory, Forbes; labourinhibitor, Theratechnolo; glaucoma therapy, Theratechnolo; AG-EM-0040;MS therapy, AplaGen; interleukin-2 mimetic, AplaGen; CNS therapy,AplaGen; Mesd-based peptides, Raptor; paratohormone, Sidus; asthmatherapy, Synairgen; dekafin-1; anticancer vaccine, Ulm; BT-15; cancerimaging agent, Speci; cardiovascular imaging, Speci; E-75; Prothyx;anticancer, Prothyx, Stealthyx; IL12-NGR; allergy vaccine, China Bio;amylin mimetic, 2nd-gen, Amylin; influenza vaccines, Variation;VLP-0012M; PLT-101; AL-408; anticancers, Aileron; antivirals, Ambrx;hSPN-16; HDL, Cerenis; enterostatin; BSc-2118; SB-006; antimicrobials,Spider Biotech; peptide therapy, Angioblast; octreotide, Ambrilia;GAP-134; Alzheimer's therapy, Il Dong; BL-4020; von Willebrand factor,Baxter; IL-1 aQb; POT-4; gamma-secretase inhibitors, BMS; ISCOMATRIX;enfuvirtide, needle-free; connexin modulators, NeuroSol; BT-25; BT-20;AmpTide; HepTide; antimicrobial peptides, Helix; NPY² agonists, Bayer;ragweed PAD, Circassia; dust mite PAD, Circassia; grass PAD, Circassia;transplant rejection PAD; insulin, oral, Oramed; cardiac ischaemiatherapy, Phy; PYC-18; antidiabetics, Phylogica; PEP-35; ACE-041;ACE-031; ovarian cancer vaccine, Generex; ATX-MS-1467; iATX FVIII;diabetes vaccine, Apitope; allergy vaccine, Apitope; FX-06 analogue;PR-22G; PR-21, Pharmaxon; LT-1945; LT-1942; XG-414; XG-517; AC-163794;MDPTQ; B27PD; AC-2307; sedatives, ProTherapeutics; L-Type Ca channelblocker, Pro; phospholipase A2 inhibitor, Pro; PGL-3001; PGL-1001;influenza vaccine, Variation-2; Homspera nanoparticle, Immune; CVX-096;COR-1; survivin-2B; imMucin; GLP-1, PharmaiN; atherosclerosis vaccine,Affir; adeptide; somatostatin antagonists, Preg; Casimax; CD-NP, Nile;PRX-111; ACT1-C; PRX-102; ACT1-G; AIDS vaccine, ITS; influenza vaccine,ITS; hepatitis-C vaccine, ITS; ALTY-0601; BGLP-40; somatropin, INB;trypansomal vaccine, INB; RU-COH, Pantarhei; LH-COH, Pantarhei; GLP-1analogues, Unigene; Polyfensin; VIR-576; Xen-0568; Xen-0495; Xen-0468;LEKTI-6; leukaemia vaccine, MD Anderson; Met receptor agonists, MRCT;insulin HDV, short-acting, Dia; glucagon antagonists, CoGene; GLP-1agonists, CoGenesys; insulin HDV, oral, Diasome; insulin HDV,long-acting, Dia; glucagon, Particle Therapeutics; GLP-1, Mannkind;insulin, next-generation, Flamel; Ostabolin-C, topical; DAC: HIV;antiviral, HepTide; Insulin Aspart, biphasic-3, No; Innotide; influenzavaccine, Bionor; HPV vaccine, Bionor Immuno; hepatitis-C vaccine,Bionor; Affitope AD-02; Affitope AD-03; RHS-02; RHS-03; insulin, Access;inherbins, Enkam; Dekafin-2; BL-4050; ALS vaccine, Amorfix; cancervaccine, Canopus; relaxin, Corthera; rhNRG-1; rhErbB3-f; hepatitis-Cvacc, Green Cross-3; androgen receptor antag, CRT; GLP-1 analogue CR,OctoPlus; AIDS vaccine, Sanofi Past-12; insulin, Diabetology; Combulin;AIDS vaccine, Sanofi Past-11; AnergiX.MG; AnergiX.MS; insulin,CritiTech; YP-20; NDR/NCE-18; CLT-002; CLT-007; CLT-008, Charlesson;CLT-009; PYC-38; AIM-101; AIM-102; AIM-501; APL-180; metabolic diseasetherapy, Xen; NP-213; NP-339; antimicrobial peptides, NovaB; lunganti-infectives, NovaBiot; c-peptide analogue, Diabetology; CGEN-855;NN-1250; N,N-9535; insulin, rectal, Oramed; insulin, 12 hr, Altea;pancreatic cancer vaccine, Onco; SB-101; L-glutamine, Emmaus; glucagonantagonists, Kisspeptin-54; Kisspeptin-14; Kisspeptin-13; Kisspeptin-10;Ziconotide; Biphalin; Nesiritide; Protegrin-1; Protegrin-2; Protegrin-3;Protegrin-4; Protegrin-5; Preprotegrin; V681; V681 (V13A_(D)); GLP-2;GLP-2 (A2G); GLP-2 (A2G/C34); AOD-9604; Ac-AOD-9604(S8K);Ac-AOD-9604(K17); C-peptide; CR845; and Marcadia.

In certain embodiments of the invention, PEP is a therapeutic peptideselected from the therapeutic peptides listed in Table 1.

Table 1

This table lists the SEQ ID NOs., names, sequences, and known orsuspected therapeutic activities of various peptides described herein.The SEQ ID NOs. 1-301 describe sequences that are required to beprovided with the Sequence Listing and are therefore appended with theinstant Specification. In some instances, these peptides containfeatures that are either inconsistent with or not amenable to inclusionin the Sequence Listing. For example, a sequence with less thanfour-amino acids; a sequence with a D-amino acid; or certainmodification that cannot be described in the Sequence Listing presently,and therefore are not provided in the Sequence Listing. However, for theease of use and description, a SEQ ID NO. has been provided to thesepeptides (i.e., SEQ ID NOs: 302-469).

(—NH₂ indicates amidation at the C-terminus; Ac indicates acetylation;other modifications are as described herein and in the specification;SIN indicates Sequence Identification Number)

SEQ ID Sequence and/or other Identifying Therapeutic NO: Name FamilyInformation Activity   1 carperitide ANP SLRRSSCFGGRMDRIGAQSGLGCNSFRY;Cardiostimulant human alpha-atrial natriuretic peptide; RespiratoryAtriopeptin-28 (human);   2 alpha- EndorphinH-Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys-OH Analgesic, otherneoendorphin   3 A-3847 Insulin gi|386828|gb|AAA59172.1|insulin [HomoAntidiabetic sapiens] MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQK RGIVEQCCTSICSLYQLENYCN   4A-4114 Insulin MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCG AntidiabeticERGFFYTPKTRREAEDLQVGQVELGG   5 A-68552GPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN Anorectic/ Antiobesity 302 andA-75998 [Ac-D-2Nal1-D-4ClPhe2-D-3Pal3-NMeTyr5-D- Releasing 303Lys(Nic)6-Lys(Isp)8-D-Ala10]GnRH; N-acetyl- hormoneD-2-naphthylalanyl-D-4-chlorophenylalanyl- Reproductive/D-3-pyridylalanyl-seryl-N-methyltyrosyl- gonadal, generalD-N(epsilon)-nicotinyllysyl-leucyl-N(epsilon)-isopropyllysyl-prolyl-alaninamide acetate   6 AN-1792 beta-gi|8176733|gb|AAB26264.2| beta-amyloid Cognition amyloidpeptide precursor; beta APP [Homo sapiens] enhancer peptideGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIIITLVMLKKQYTSNHHGVVE   7 AAMP-1MESESESGAAADTPPLETLSFHGDEEIIEVVELDPGPPDPDDLA AnticoagulantQEMEDVDFEEEEEEEGNEEGWVLEPQEGVVGSMEGPDDSEVTFA Anti-LHSASVFCVSLDPKTNTLAVTGGEDDKAFVWRLSDGESSFECAG inflammatoryHKDSVTCAGFSHDSTLVATGDMSGLLKVWQVDTKEEVWSFEAGD ImmunologicalLEWMEWHPRAPVLLAGTADGNTWMWKVPANGDCKTFQGPNCPAT Anticancer,CGRVLPDGKRAVVGYEDGTIRIWDLKQGSPIHVLKGTEGHQGPL otherTCVAANQDGSLILTGSVDCQAKLVSATTGKVVGVFRPETVASQP VulnerarySLGEGEESESNSVESLGFCSVMPLAAVGYLDGTLAIYDLATQTLRHQCQHQSGIVQLLWEAGTAVVYTCSLDGIVRLWDARTGRLLTDYRGHTAEILDFALSKDASLVVTTSGDHKAKVFCVQRPDR   8 Exenatide GLP-1HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Antidiabetic Anorectic/Antiobesity   9 AC-625 Acetyl-ATQRLANELVRLQTYPRTNVGSNTY-NH₂ Anti-hypertensive, renin system Symptomatic antidiabetic  10 ACTHgi|80861463|ref|NP_001030333.1| Adrenal andproopiomelanocortin preproprotein pituitary [Homo sapiens] disordersMPRSCCSRSGALLLALLLQASMEVRGWCLESSQCQDLTTESNLLECIRACKPDLSAETPMFPGNGDEQPLTENPRKYVMGHFRWDRFGRRNSSSSGSSGAGQKREDVSAGEDCGPLPEGGPEPRSDGAKPGPREGKRSYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEFKRELTGQRLREGDGPDGPADDGAGAQADLEHSLLVAAEKKDEGPYRMEHFRWGSPPKDKRYGGFMTSEKSQTPLVTLFKNAIIKNAYK KGE  11 AIDSgi|288842|emb|CAA78890.1| V3 loop Therapeutic therapeutic[Human immunodeficiency virus type 1] vaccine vaccineCTRPSNNTRKSIPVGPGKALYATGAIIGNIRQAHC  12 AIDS therapygi|5081475|gb|AAD39400.1|AF128998_1 gag Antiviral,[Human immunodeficiency virus type 1] anti-HIVMGARASVLSGGKLDKWEKIRLRPGGKKTYQLKHIVWASRELERFAVNPGLLETGGGCKQILVQLQPSLQTGSEELKSLYNAVATLYCVHQGIEVRDTKEALDKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPAHAGPNAPGQMREPRGSDIAGTTSTLQEQIGWMTSNPPVPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPSHKARILAEAMSQVTSPANIMMQRGNFRNQRKTIKCFNCGKEGHLARHCRAPRKKGCWKCGREGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPPQKQEPLPSQKQ ETIDKDLYPLASLKSLFGNDPSLQ13 and 14 Allotrap-2702 Allotrap 1258; Allotrap 2702; Allotrap Immuno-E; Allotrap G; RDP58; peptide Bc-1nl; suppressant NLRIALR/RLAIRLN15 and 16 Alzheimer's H-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-Imaging agent imaging agent OH; or H-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-OH  17 AM-425 gi|4504991|ref|NP_002300.1| leukemiaAntiarthritic, inhibitory factor (cholinergic immunologicaldifferentiation factor) [Homo sapiens]MKVLAAGVVPLLLVLHWKHGAGSPLPITPVNATCAIRHPCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPNNLDKLCGPNVTDFPPFHANGTEKAKLVELYRIVVYLGTSLGNITRDQKILNPSALSLHSKLNATADILRGLLSNVLCRLCSKYHVGHVDVTYGPDTSGKD VFQKKKLGCQLLGKYKQIIAVLAQAF304 AN-238 L-Threoninamide, N-[5-[2-[(2S,4S)- Somatostatin1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy- Anticancer,7-methoxy-6,11-dioxo-4-[[2,3,6-trideoxy- hormonal3-(2,3-dihydro-1H-pyrrol-1-yl)-alpha-L-lyxo-hexopyranosyl]oxy]-2-naphthacenyl]-2-oxoethoxy]-1,5-dioxopentyl]-D-phenylalanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-, cyclic (2-7)-disulfide 305 AV-9 [D-Arg]9-NH₂Antiviral, other   8 AZM-134 GLP-1HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Anorectic/ AntiobesityAntidiabetic  18 Addressin gi|109633022|ref|NP_570116.2| mucosalRecombinant, vascular addressin cell adhesion molecule other1 isoform a precursor [Homo sapiens] Anti-MDFGLALLLAGLLGLLLGQSLQVKPLQVEPPEPVVAVALGASRQ inflammatoryLTCRLACADRGASVQWRGLDTSLGAVQSDTGRSVLTVVYAFPDRNASLSAAGTRVCVGSCGGRTFQHTVQLLQLTVSPAALVPGDPEVACTAHKVTPVDPNALSFSLLVGGQELEGAQALGPEVQEEEEEPQGDEDVLFRVTERWRLPPLGTPVPPALYCQATMRLPGLELSHRQAIPVLHSPTSPEPPDTTSPESPDTTSPESPDTTSQEPPDTTSPEPPDKTSPEPAPQQGSTHTPRSPGSTRTRRPEISQAGPTQGEVIPTGSSKPAGDQLPAALWTSSAVLGLLLLALPTYHLWKRCRHLAEDDTHPPASLRLLPQVSAWAGLRGTGQVGISPS 306 ambamustineL-Methionine, N-[3-[bis(2-chloroethyl)amino]- Anticancer,N-(4-fluoro-L-phenylalanyl)-L-phenylalanyl]-, alkylating ethyl esterAnticancer, antimetabolite  19 amylinDTTVSEPAPSCVTLYQSWRYSQADNGCAETVTVKVVYEDDTEGL Antidiabetic antagonistsCYAVAPGQITTVGDGYIGSHGHARYLARCL  20 anaritide ANPgi|178638|gb|AAA35529.1| atrial Anti- analogues natriuretic peptidehypertensive, MSSFSTTTVSFLLLLAFQLLGQTRANPMYNAVSNADLMDFKNLL diureticDHLEEKMPLEDEVVPPQVLSDPNEEAGAALSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRSALLKSKLRALLTAPRSLRRSSCFG GRMDRIGAQSGLGCNSFRY 21-28anti- As disclosed in U.S. Pat. No. 5,470,831: Anti- inflammatoryThr-Thr-Ser-Gln-Val-Arg-Pro-Arg inflammatory peptideVal-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg. Immuno- Ser-Gln-Val-Arg-Pro-Argsuppressant Val-Arg-Pro-Arg MultipleThr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr. sclerosisThr-Thr-Ser-Gln-Val treatment Thr-Ser-Gln-Val-Arg Antiarthritic,Thr-Thr-Ser-Gly-Ile-His-Pro-Lys other Stomatological Dermatological 307antiflammins L-Leucine, N-[N-[N-[N-[N2-[N2-[N-(N-L- Anti-histidyl-L-alpha-aspartyl)-L-methionyl]-L- inflammatoryasparaginyl]-L-lysyl]-L-valyl]-L-leucyl]-L- alpha-aspartyl]- 308antifungal tripeptides of N3-4-methoxyfumanyl and di-  Antifungaltripeptides and tripeptides of N3-D-trans 2,3-epoxysuccinamoyl-L-2,3-diaminopropanoic acid  29 GastrimmuneG17-DT; G17DT (vaccine); Gastrimmune; Anticancer,Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu- immunologicaldiphtheria toxoid; anti-gastrin 17 immunogen;gastrin 17 vaccine; gastrin-17-diphtheria toxoid conjugate  30antithrombin gi|312673|emb|CAA51292.1| Hirudin Antithromboticpolypeptides [Hirudinaria manillensis] AnticoagulantMFSLKLFVVFLAVCICVSQAVSYTDCTESGQNYCLCVGGNLCGGGKHCEMDGSGNKCVDGEGTPKPKSQTEGDFEEIPDEDILN  31 antiviralNH₂-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-COOH Antiviral, other peptides 32 apolipoprotein gi|671882|emb|CAA28583.1| apolipoproteinHypolipaemic/ [Homo sapiens] Antiathero-MKLLAATVLLLTICSLEGALVRRQAKEPCVESLVSQYFQTVTDY sclerosisGKDLMEKVKSPELQAEAKSYFEKSKEQLTPLIKKAGTELVNFLS YFVELGTHPATQ  33 arthritisgi|46369603|gb|AAS89650.1| secreted antigen Recombinant, antigen85A precursor [Mycobacterium bovis BCG] otherMQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAF Antiarthritic,SRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQ immunologicalDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGK Immuno-AGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSAL suppressantTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA 309 Avorelin5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L- Releasingseryl-L-tyrosyl-2-methyl-D-tryptophyl-L- hormoneleucyl-L-arginyl-N-ethyl-L-prolinamide Anticancer, hormonal Menstruationdisorders 310 B-956 N-[8(R)-Amino-2(S)-benzyl-5(S)-isopropyl-9-Anticancer, sulfanyl-3(Z),6(E)-nonadienoyl]-L-methionine other 311BCH-2687 L-Tyrosyl-D-arginyl-L-phenylalanyl-L- Analgesic, otherphenylalaninamide  34 BCH-2763 L-Leucine, D-phenylalanyl-L-prolyl-5-Antithrombotic aminopentanoyl-5-aminopentanoyl-L-alpha- Anticoagulantaspartyl-L-phenylalanyl-L-alpha-glutamyl-L-prolyl-L-isoleucyl-L-prolyl-; BCH-2763;Phe-Pro-(NH(CH₂)₄CO)₂-Asp-Phe-Glu-Pro-Ile-Pro-Leu; phenylalanyl-prolyl-(NH(CH₂)₄CO)₂-aspartyl-phenylalanyl-glutamyl-prolyl- isoleucyl-prolyl-leucine 312frakefamide L-phenylalaninamide, L-tyrosyl-D-alanyl-4- Analgesic, otherfluoro-L-phenylalanyl- 313 BIM-22015Glycinamide, D-alanyl-L-glutaminyl-L-tyrosyl- ACTHL-phenylalanyl-L-arginyl-L-tryptophyl- Neurological  35 BIM-26028Pyroglutamyl-glutaminyl-arginyl-leucyl- Releasingglycyl-asparaginyl-glutaminyl-tryptyl-alanyl- hormonevalyl-glycyl-histidinyl-leucyl-leucyl-NH₂ Respiratory Anorectic/Antiobesity Anticancer, hormonal 314 BIM-44002L-Tyrosinammide, L-phenylalanyl-L-norleucyl- HormoneL-histidyl-L-asparaginyl-L-leucyl-D- Osteoporosistryptophyl-L-lysyl-L-histidyl-L-leucyl-L- treatmentseryl-L-seryl-L-norleucyl-L-alpha-glutamyl-L-arginyl-L-valy-L-.alpha.-glutamyl-L-tryptophyl-L-leucyl-L-arginyl-L-lysyl-L-lysyl-L-leucyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidyl-L-asparaginyl-  36 BIO-1211 L-Proline, N-((4-((((2-Antiasthma methylphenyl)aminocarbonyl)amino) GI inflamma-phenyl)acetyl)-L-leucyl-Lalpha- tory/bowelaspartyl-L-valyl-; BIO-1211; N-((4- disorders((((2-methylphenyl)amino)carbonyl) Multipleamino)phenyl)acetyl)-leucyl-aspartyl- sclerosis valyl-proline treatment 37 BPC-15 BPC 15; BPC-15; BPC-157; booly protection Anti-compound 15; L-Valine, glycyl-L-alpha- inflammatoryglutamyl-L-prolyl-L-prolyl-L-prolylglycyl-L-lysyl-L-prolyl-L-alanyl-L-alpha-aspartyl-L-alpha-aspartyl-L-alanylglycyl-L-leucyl- 315 bivalirudinL-Leucine, D-phenylalanyl-L-prolyl-L- Anticoagulantarginyl-L-prolylglycylglycylglycylgylcyl- AntianginalL-asparaginylglycl-L-alpha-aspartyl-L-phenylalanyl-L-alpha-glutamyl-L-alpha-glutamyl-L-isoleucyl-L-prolyl-L-alpha- glutamyl-L-tyrosyl-;D-phenylalanyl-L-prolyl-L-arginyl-L-prolyl-glycylglycyl-glycyl-gylcyl-L-asparagyl-glycyl-L-aspartyl-L-phenylalanyl-L-glutamyl-L-glutamyl-L-isoleucyl-Lprolyl-L-glutamyl-L-glutamyl-L-tyrosyl-L-leucine trifluoroacetate (salt) hydrate  38bombesin 5-oxoPro-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val- Anticancer,antagonist Gly-His-Leu-MetNH₂ [CAS], otherBombesin 14; Bombesin Dihydrochloride; Dihydrochloride, Bombesin  39brain BNP SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH COPD treatment, natriureticcardiac peptide  41 C-peptide C-peptideGlu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu- Symptomatic analoguesLeu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln- antidiabeticPro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln Ophthalmological Neurological 316C5a antagonist Me-Phe-Lys-Pro-D-Cha-L-Cha-D-Phe Anti- inflammatory  42CBT-101 L-Cysteinamide, L-asparaginyl-L-leucylglycyl- AntiglaucomaL-valyl-S-[(acetylamino)methyl]-, monoacetate  43 CCK(27-32)Tyr(SO₃)-Met-Gly-Trp-Met-Asp; CBZ-CCK Analgesic,(27-32)-NH₂; cholecystokinin (27-32) amide, obesity, otherbenzoyloxycarbonyl-, D-Trp  44 CD4CD4 (81-92), D-Ile; CD4 (81-92), D-Tyr; Antiviral, anti-CD4 (81-92), D-Tyr,D-Cys,D-Glu(5); HIVCD4(81-92); TYICEVEDQKEE; Thr-Tyr-Ile-Cys-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu; threonyl-tyrosyl-isoleucyl-cysteinyl-glutamyl-valyl-glutamyl-aspartyl-flutaminyl-lysyl-glutamyl- glutamic acid 317CEE-04-420 Lys-D-Pro-Thr and Lys-D-Pro-Val Analgesic, other  45 CEP-079gi|108796063|ref|NP_001007140.2| insulin- Ophthalmologicallike growth factor 2 isoform 1 precursor [Homo sapiens]MGIPMGKSMLVLLTFLAFASCCIAAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSERDVSTPPTVLPDNFPRYPVGKFFQYDTWKQSTQRLRRGLPALLRARRGHVLAKELEAFREAKRHRPLIALPTQDPAHGGAPPEMA SNRK 318 mifamurtideL-Alaninamide, N-(N-acetylmuramoyl)-L-alanyl- Anticancer,D-alpha-glutaminyl-N-[4-hydroxy-10-oxo-7- immunological[(1-oxohexadecyl)oxy]-3,5,9-trioxa-4-phosphapentacos-1-yl]-, P-oxide, monosodium salt, (R)-  46 CGRP CGRPACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH₂ Hormone analoguesCardiovascular  47 rusalatide gi|4503635|ref|NP_000497.1| coagulation Musculoskeletal acetate factor II prepropretein [Homo sapiens] VulneraryMAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRA SymptomaticNTFLEEVRKGNLERECVEETCSYEEAFEALESSTATDVFWAKYT antidiabeticACETARTPRDKLAACLEGNCAEGLGTNYRGHVNITRSGIECQLW CardiovascularRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTV Anti-infective,RRQECSIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQ otherYQGRLAVTTHGLPCLAWASAQAKALSKHQDFNSAVQLVENFCRN OphthalmologicalPDGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRAIEGRTATSEYQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIEKISMLEKIYIHPRYNWRENLDRDIALMKLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGEGCDRDGKYGFYTHVFRLKKWIQKV IDQFGE  48 CKS-17L-Leucine, L-leucyl-L-glutaminyl-L- Immuno-asparaginyl-L-arginyl-L-arginylglycyl-L- suppressantleucyl-L-alpha-aspartyl-L-leucyl-L-leucyl-L- Anticancer,phenylalanyl-L-leucyl-L-lysyl-L-alpha- immunologicalglutamylglycylglycyl-; CKS-17; CKS-17 peptide  10 corticorelin cortico-gi|80861463|ref|NP_001030333.1| Neuroprotective acetate tropinproopiomelanocortin preproprotein Antiasthma [Homo sapiens] Anti-MPRSCCSRSGALLLALLLQASMEVRGWCLESSQCQDLTTESNLL inflammatoryECIRACKPDLSAETPMFPGNGDEQPLTENPRKYVMGHFRWDRFGRRNSSSSGSSGAGQKREDVSAGEDCGPLPEGGPEPRSDGAKPGPREGKRSYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEFKRELTGQRLREGDGPDGPADDGAGAQADLEHSLLVAAEKKDEGPYRMEHFRWGSPPKDKRYGGFMTSEKSQTPLVTLFKNAIIKNAYK KGE  49 CT-112L-Arginine, L-threonyl-L-threonyl-L-seryl-L- Antiarthritic,glutaminyl-L-valyl-L-arginyl-L-prolyl-; immunological5-(3-ethoxy-4-pentyloxyphenyl)-2,4- thiazolidinedione  50 CTAP-IIIphenylalanyl--cysteinyl--tyrosyl-tryptophyl- Vulneraryarginyl-threonyl-penicillaminyl- Antiarthritic,threoninamide; rCTAP-III-Leu-21 (des 1-15); othersomatostatin analog CTAP Musculoskeletal Recombinant, other  51 CVFMCys-Val-Phe-Met Anticancer, other 52 and 53 calcitonin calci-CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP (human) Formulation, toninH-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly- oral, otherLys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr- HormoneTyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro- Osteoporosis NH₂ (salmon)treatment  54 calciseptine sp|P22947|TXCAS_DENPO Calciseptin OS = Anti-Dendroaspis polylepis polylepis PE = 1 hypertensive, SV = 1 otherRICYIHKASLPRATKTCVENTCYKMFIRTQREYISERGCGCPTA MWPYQTECCKGDRCNK 52 and 53calcitonin calci- CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP (human) Hormoneanalogues tonin H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly- OsteoporosisLys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr- treatmentTyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro- NH₂ (salmon)  55calphobindin I gi|186680508|ref|NM_001154.3| Homo sapiens Ophthalmo-annexin A5 (ANXA5), logical,MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTS VulneraryRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDY KKALLLLCGEDD 319 cargutocin1,6-Dicarbaoxytocin, 1-butanoic acid-7- Labour inducer glycine- 320casokefamide L-Tyrosinamide, L-tyrosyl-D-alanyl-L- Antidiarrhoealphenylalanyl-D-alanyl-  56 cekropin-P sp|P14661|CECP1_PIG Cecropin-P1Antibacterial, OS = Sus scrofa PE = 1 SV = 1 otherSWLSKTAKKLENSAKKRISEGIAIAIQGGPR  57 tasidotinN,N-Dimethyl-L-valyl-L-valyl-N-methyl- Anticancer, hydrochlorideL-valyl-L-propyl-L-proline-tert-butylamide other  58 ceruletidePyr-Gln-Asp-Tyr(SO3H)-Thr-Gly-Trp-Met-Asp- Analgesic, other diethylaminePhe-C(O)—NH₂ Gastroprokinetic 321 cetrorelixD-Alaninamide, N-acetyl-3-(2-naphthalenyl)- Fertility acetateD-alanyl-4-chloro-D-phenylalanyl-3-(3- enhancerpyridinyl)-D-alanyl-L-seryl-L-tyrosyl-N5- Prostate(aminocarbonyl)-D-ol-L-leucyl-L-arginyl- disorders L-prolyl-Menstruation disorders Anticancer, hormonal  59 corticoliberin cortico-SQEPPISLDLTFHLLREVLEMTKADQLAQQAHSNRKLLDIA Releasing liberin hormone 322D-22213 L-Histidinamide, N2-[(2,3,4,9-tetrahydro-1H- Anticancer,pyrido[3,4-b]indol-3-yl)carbonyl]-L- otherglutaminyl-L-tryptophyl-L-alanyl-L-valylglycyl-N-[1-[[[1-(aminocarbonyl)-3-methylbutyl]amino]methyl]-3-methylbutyl]-,[1(R),6[S-(R*,R*)]]-, monoacetate 323 DAP inhibitorsL-AP-L-Ala and L-Ala-L-Ala-DL-AP; Antibacterial, other  60 DP-640insulin L-Tyrosinamide, β-alanyl-L-arginylglycyl-L- Insulinphenylalanyl-L-phenylalanyl-, diacetate Antidiabetic (salt)  61 DP-107L-Leucine, L-methionyl-L-threonyl-L-leucyl- Antiviral,L-threonyl-L-valyl-L-glutaminyl-L-alanyl-L- anti-HIVarginyl-L-glutaminyl-L-leucyl-L-leucyl-L-seryl-L-glutaminyl-L-isoleucyl-L-valyl-L-glutaminyl-L-glutaminyl-L-glutaminyl-L-asparaginyl-L-asparaginyl-L-leucyl-L-leucyl-L-arginyl-L-alanyl-L-isoleucyl-L-.alpha.-glutamyl-L-alanyl-L-glutaminyl-L-glutaminyl-L-histidyl-L-leucyl-L-leucyl-L-glutaminyl-L-leucyl-L-threonyl-L-valyl-L- tryptophylglycyl-L-isoleucyl-L-lysyl-L-glutaminyl-  62 DU-728 Arg-Gly-Asp-Ser Antithrombotic  63 Dynorphin AH-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro- Analgesic, otherLys-Leu-Lys-Trp-Asp-Asn-Gln-OH Neuroprotective Dependence treatment  64defensins gi|181535|gb|AAA52304.1| defensin precursor Antibiotic,MRTLAILAAILLVALQAQAEPLQARADEVAAAPEQIAADIPEVV otherVSLAWDESLAPKHPGSRKNMDCYCRIPACIAGERRYGTCIYQGR Antifungal LWAFCC Vulnerary324 detirelix D-Alaninamide, N-acetyl-3-(2-naphthalenyl)- ReleasingD-alanyl-4-chloro-D-phenylalanyl-D- hormonetryptophyl-L-seryl-L-tyrosyl-N6- Abortifacient[bis(ethylamino)methylene]-D-lysyl-L-leucyl- Male L-arginyl-L-prolyl-contraceptive  65 disagregin gi|545738|gb|AAB30092.1| disagregin =Antithrombotic fibrinogen receptor antagonist [OrnithodorosCardiovascular moubata = tick, salivary gland, Peptide, 60 aa]SDDKCQGRPMYGCREDDDSVFGWTYDSNHGQCWKGSYCKHRRQP SNYFASQQECRNTCGA 66 and 65E-2078 D-Leucinamide, N-methyl-L- Analgesic,tyrosylglycylglycyl-L-phenylalanyl-L-leucyl- otherL-arginyl-N2-methyl-L-arginyl-N-ethyl-SDDKCQGRPMYGCREDDDSVFGWTYDSNHGQCWKGSYCKHRRQP SNYFASQQECRNTCGA ELS-1Arg-Lys-Glu Immunostimulant, other  67 ecallantideGlu-Ala-Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala- Angiodema, Anti-Asp-Asp-Gly-Pro-Cys-Arg-Ala-Ala-His-Pro-Arg- inflammatory,Trp-Phe-Phe-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Glu- Haemostatic,Glu-Phe-Ile-Tyr-Gly-Gly-Cys-Glu-Gly-Asn-Gln- Antiarthritic,Asn-Arg-Phe-Glu-Ser-Leu-Glu-Glu-Cys-Lys-Lys- other Met-Cys-Thr-Arg-Asp325 ES-1005 bis-(1-naphthyl)methylacetyl-His-Sta- Anti- Leu-E-Lys diHClhypertensive, renin system 326 efegatranL-prolinamide, N-methyl-D-phenylalanyl-n-(4- Antithrombotic((aminoiminomethyl)amino)-1-formylbutyl), Antianginal (S)- 68 and 69elafin gi|999146|gb|AAB34627.1| elafin [Homo Respiratory derivativessapiens] COPD treatment MRASSFLIVVVFLIAGTLVLE Antiarthritic,H-Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-Val-Ser- otherThr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala-Met-Leu-Asn-Pro-Pro-Asn-Arg-Cys-Leu-Lys-Asp-Thr-Asp-Cys-Pro-Gly-Ile-Lys-Lys-Cys-Cys-Glu-Gly-Ser-Cys-Gly-Met-Ala-Cys-Phe-Val-Pro-Gln-OH (Disulfide bonds betweenCys16-Cys45, Cys23-Cys49, Cys32-Cys44, Cys38-Cys53) 70 and 52 elcatonincalci- Ser-Asn-Leu-Ser-Thr-Asn-Val-Leu-Gly-Lys-Leu- Hormone toninSer-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro- OsteoporosisArg-Thr-Asn-Val-Gly-Ala-Gly-Thr-Pro-NH₂ treatmentCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP (human) Analgesic, other  71 eledoisin5-oxo-L-Pro-L-Pro-L-Ser-L-Lys-L-Asp-L-Ala-L- OphthalmologicalPhe-L-Ala-L-isoleucylglycyl-L-Leu-L- methionin-amide   3 encapsulatedinsulin gi|386828|gb|AAA59172.1| insulin [Homo Formulation, insulinsapiens] optimized, MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGnanoparticles ERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQK InsulinRGIVEQCCTSICSLYQLENYCN Antidiabetic  72 endorphin, β-YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE Analgesic, other  72 endorphin,YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE Analgesic, other pancreatic  73endothelial cell gi|189701|gb|AAA60043.1| endothelial Cardiovasculargrowth factor cell growth factorMAALMTPGTGAPPAPGDFSGEGSQGLPDPSPEPKQLPELIRMKRDGGRLSEADIRGFVAAVVNGSAQGAQIGAMLMAIRLRGMDLEETSVLTQALAQSGQQLEWPEAWRQQLVDKHSTGGVGDKVSLVLAPALAACGCKVPMISGRGLGHTGGTLDKLESIPGFNVIQSPEQMQVLLDQAGCCIVGQSEQLVPADGILYAARDVTATVDSLPLITASILSKKLVEGLSALVVDVKFGGAAVFPNQEQARELAKTLVGVGASLGLRVAAALTAMDKPLGRCVGHALEVEEALLCMDGAGPPDLRDLVTTLGGALLWLSGHAGTQAQGAARVAAALDDGSALGRFERMLAAQGVDPGLARALCSGSPAERRQLLPRAREQEELLAPADGTVELVRALPLALVLHELGAGRSRAGEPLRLGVGAELLVDVGQRLRRGTPWLRVHRDGPALSGPQSRALQEALVLSDRAPFAAPSPFAELVLPPQQ  74 eptifibatideMAP-HAR-GLY-ASP-TRP-PRO-CYS-NH₂ Antianginal Cardiovascular 327examorelin GHRP L-Lysinamide, L-histidyl-2-methyl-D- Releasingtryptophyl-L-alanyl-L-tryptophyl-D- hormone phenylalanyl- VulneraryCardiovascular  75 FG-005 SMR1-QHNPR Male sexual dysfunction 328FR-113680 L-Phenylalaninamide, N-acetyl-L-threonyl-1- Antiasthmaformyl-D-tryptophyl-N-methyl-N- (phenylmethyl)-  76 fibronectin-Gly-Arg-Gly-Asp-Ser Anticancer, related peptide other 329 G-4120L-Cysteine, N-(mercaptoacetyl)-D-tyrosyl-L- Antithromboticarginylglycyl-L-alpha-aspartyl-,  cyclic (1-5)-sulfide, S-oxide 330EP-51216 2S)-6-amino-2-[[(2S)-2-[[(2R)-2-[[(2R)-2-(4- GH Releasingaminobutanoylamino)-3-(2-methyl-1H-indol-3- hormoneyl)propanoyl]amino]-3-(2-methyl-1H-indol-3- Vulnerary,yl)propanoyl]amino]-3-(2-methyl-1H-indol-3- endocrineyl)propanoyl]amino]hexanamide   8 GLP-1 + GLP-1HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Antidiabetic exendin-4  77GM-1986 YYWIGIR Anti- inflammatory  78 GnRH- GnRHgi|133908612|ref|NP_001076580.1| Antiprolactin associatedgonadotropin-releasing hormone 1 precursor Menstruation peptide[Homo sapiens] disorders MKPIQKLLAGLILLTWCVEGCSSQHWSYGLRPGGKRDAENLIDSFertility FQEIVKEVGQLAETQRFECTTHQPRSPLRDLKGALESLIEEETG enhancer QKKI  79GRF1-44 gi|11034841|ref|NP_066567.1| growth hormone Musculoskeletalreleasing hormone preporprotein [Homo sapiens]MPLWVFFFVILTLSNSSHCSPPPPLTLRMRRYADAIFTNSYRKV  80 GRF GHRFgi|337133|gb|AAA52609.1| growth hormone Idiopathic releasing factorgrowth MPLWVFFFVILTLSNSSHCSPPPPLTLRMRRYADAIFTNSYRKV hormoneLGQLSARKLLQDIMSRQQGESNQERGARARLGRQVDSMWAEQKQ deficiency;MELESILVALLQKHRNSQG cachexia 331 GYKI-14451 L-Prolinamide, N-[(1,1-Antithrombotic dimethylethoxy)carbonyl]-D-phenylalanyl-N-[4-[(aminoiminomethyl)amino]-1- formylbutyl]-, (S)-  81 galaningi|1247490|emb|CAA01907.1| galanin Releasing [Homo sapiens] hormoneMARGSALLLASLLLAAALSASAGLWSPAKEKRGWTLNSAGYLLGPHAVGNHRSFSDKNGLTSKRELRPEDDMKPGSFDRSIPENNIMRTIIEFLSFLHLKEAGALDRLLDLPAAASSEDIERS  82 gastrin(benzyloxycarbonyl)-L-Glu-L-Ala-L-Tyr- Antiulcer antagonistsGly-L-Tyr-L-Met-L-aspartic acid amide 332 glaspimodN2,N2′-[2,7-Bis(pyroglutamyl-glutamyl- Immunomodulator,aspartylamino)-octanediolyl]bis(lysine) anti-infective Immunostimulant,other Radio/chemo- protective  83 glicentingi|125987831|sp|P01275.3|GLUC_HUMAN Glucagon Insulinprecursor [Contains: Glicentin; Glicentin- Antiulcerrelated polypeptide (GRPP); Oxyntomodulin Antidiabetic(OXY) (OXM); Glucagon; Glucagon-like peptide 1 (GLP-1); Glucagon-likepeptide 1(7-37) (GLP-1(7-37));Glucagon-like peptide 1(7-36) (GLP-1(7-36));Glucagon-like peptide 2 (GLP-2)]MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSDYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKI TDRK  84 glucagonH₂N-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- hypoglycemiaSer-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp- DiagnosticPhe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH  84 glucagon glucagonHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- HypoglycemiaLys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe- Val-Gln-Trp-Leu-Met-Asn-Thr 85 gonadorelin gonado- gi|121522|sp|P01148.1|GON1_HUMAN Femaleanalogues relin Progonadoliberin-1 precursor contraceptive;(Progonadoliberin I) [Contains: enometiosis,Gonadoliberin-1 (Gonadoliberin I) uterine(Luteinizing hormone-releasing hormone I) leiomyoma,(LH-RH I) (Gonadotropin-releasing hormone I) precocious(GnRH-I) (Luliberin I) (Gonadorelin); GnRH- puberty,associated peptide 1 (GnRH-associated prostate and peptide I)]breast cancer MKPIQKLLAGLILLTWCVEGCSSQHWSYGLRPGGKRDAENLIDSFQEIVKEVGQLAETQRFECTTHQPRSPLRDLKGALESLIEEETG QKKI 333 gonadorelin[Ac-DNAL1(2),4FDPhe2,D-Trp3,D-Arg6]-LHRH Female antagonistcontraceptive; enometiosis, uterine leiomyoma, precocious puberty,prostate and breast cancer  86 gonadorelin gonado-5-oxo-L-His-L-Trp-L-Ser-L-Tyr-Gly-L-Leu-L- Female relinArg-L-Pro-glycinamide contraceptive; enometiosis, uterine leiomyoma,precocious puberty, prostate and breast cancer 334 goralatideL-Proline, 1-[N2-[N-(N-acetyl-L-seryl)- HaematologicalL-α-aspartyl]-L-lysyl]- Immunological Radio/chemo- protective 335 H-142L-Lysine, N2-[N-[N-[N-[4-methyl-2-[[N-[N-[1- Anti-(N-prolyl-L-histidyl)-L-prolyl]-L- hypertensive,phenylalanyl]-L-histidyl]amino]pentyl]-L- renin systemvalyl]-L-isoleucyl]-L-histidyl]-, (S)- 336 I5B2L-Tyrosinamide, N-methyl-L-valyl-N-[2- Anti-(4-hydroxyphenyl)-1-phosphonoethyl]- hypertensive, renin system  87iseganan L-Argininamide, L-arginylglycylglycyl-L- Antibacterial,hydrochloride leucyl-L-cysteinyl-L-tyrosyl-L-cysteinyl-L- otherarginylglycyl-L-arginyl-L-phenylalanyl-L- Antifungalcysteinyl-L-valyl-L-cysteinyl-L-valylglycyl, Antiviral, othercyclic (5-14),(7-12)-bis(disulfide) hydrochloride  88 netamiftideL-Tryptophanamide, 4-fluoro-L-phenylalanyl- Antidepressant(4R)-4-hydroxy-L-prolyl-L-arginylglycyl-, Anxiolyticbis(trifluoroacetate)(salt) 337 icrocaptideL-Arginine, glycyl-N2-ethyl-L-lysyl-L- Cardiovascular prolyl-Septic shock treatment 338 icatibantL-Arginine, D-arginyl-L-arginyl-L-prolyl- Cardiovasculartrans-4-hydroxy-L-prolylglycyl-3-(2- Hepatoprotectivethienyl)-L-alanyl-L-seryl-D-1,2,3,4- Vulnerarytetrahydro-3-isoquinolinecarbanyl-L-(2α,3aβ,7aβ)-octahydro-1H-indole-2-carbonyl- 339 AG-17763-[2(S)-Hydroxy-3(S)-(3-hydroxy-2- Antiviral, anti-methylbenzamido)-4-phenylbutanoyl]-5,5- HIVdimethyl-N-(2-methylbenzyl)thiazolidine- 4(R)-carboxamide 340pralmorelin L-Lysinamide, D-alanyl-3-(2-naphthalenyl)- DiagnosticD-alanyl-L-alanyl-L-tryptophyl-D- Releasing phenylalanyl- hormone  89katacalcin calcito- gi|115478|sp|P01258.1|CALC_HUMAN CalcitoninOsteoporosis nin precursor [Contains: Calcitonin; Katacalcin treatment(Calcitonin carboxyl-terminal peptide) (CCP) Hormone (PDN-21)]Recombinant, MGFQKFSPFLALSILVLLQAGSLHAAPFRSALESSPADPATLSE otherDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRSKRCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPGKKRDMSSDLERDHRP HVSMPQAN 341ketomethylureas N-[N-[3-benzoylamino-4-phenyl-2- Anti-oxobutyl]-N-methylaminocarbonyl]proline hypertensive, renin system  90L-346670 N-L-arginyl-8-L-methoinine-21a-L- Anti-phenylalanine-21b-L-arginine-21c-L- hypertensive, tyrosine- diuretic  91L-364210 N-isovaleryl-L-histidyl-L-prolyl-L- Anti-phenylalanyl-L-histidyl-(3S,4S)-4-amino-5- hypertensive,cyclohexyl-3-hydroxypentanoic acid)-L- renin systemleucyl-L-phenylalanylamide 342 L-659837L-Phenylalanine, N-[2-(3-amino-2-oxo-1- Analgesic, otherpyrrolidinyl)-4-methyl-1-oxopentyl]-L-methionyl-L-glutaminyl-L-tryptophyl-, cyclic (4-1)-peptide, [S-(R*,S*)]-343 L-693549 5(S)-(tert-butoxycarbonylamino)-4(S)- Antiviral, anti-hydroxy-N-[2(R)-hydroxyindan-1(S)-yl]-2(R)- HIV[4-(3-hydroxypropyl)benzyl]-6-phenylhexamide 344 L-709049L-Alaninamide, N-acetyl-L-tyrosyl-L- Anti-inflmmatoryvalyl-N-(2-carboxy-1-formylethyl)-, (S)-  92 LDV-4-((N′-2-methylphenyl)ureido)phenylalanyl- Anticancer, containingleucyl-alpha-aspartyl-valyl-prolyl-alanyl- other peptides alanyl-lysineLys-Phe L-Phenylalanine, N-L-lysyl- Haematological Antisickling  93lagatide D-Alaninamide, L-prolyl-L-valyl-L- Antidiarrhoealthreonyl-L-lysyl-L-prolyl-L-glutaminyl-  94 laminin Aseryl-isoleucyl-lysyl-valyl-alanyl- Anticancer, peptide valinamide otherNeurological  95 laminin tyrosyl-isoleucyl-glycyl-serylarginineAnticancer, other 345 lanreotide somato-L-Threoninamide, 3-(2-naphthalenyl)-D- Acromegaly statinalanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl- Anticancer,L-lysyl-L-valyl-L-cysteinyl-, cyclic (2-7) hormonal-disulfide; L-Threoninamide, 3-(1- Cardiovascularnaphthalenyl)-D-alanyl-L-cysteinyl-L- Antidiarrhoealtyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-, cyclic (2-7)-disulfide 346 leuprolideLuteinizing hormone-releasing factor Formulation, acetate(pig), 6-D-leucine-9-(N-ethyl-L- implant prolinamide)-10-deglycinamide-Anticancer, hormonal Menstruation disorders 347 MCI-826Butanoic acid, 3,3-diethyl-4-[[3-[2-[4- Antiasthma (1-methylethyl)-2-thiazolyl]ethenyl]phenyl]amino]-4-oxo-, (E)-  96 omigananL-lysinamide, L-isoleucyl-L-leucyl-L- Peptide pentahydro-arginyl-L-tryptophyl-L-prolyl-L-tryptophyl- antibiotic chlorideL-tryptophyl-L-prolyl-L-tryptophyl-L-arginyl-L-arginyl, pentahydrochloride  97-100 MBPgi|68509940|ref|NP_001020272.1| Golli-mbp Multipleisoform 1 [Homo sapiens] sclerosisMGNHAGKRELNAEKASTNSETNRGESEKKRNLGELSRTTSEDNE treatmentVFGEADANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSR Immuno-PHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDSAATSESLD suppressantVMASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR gi|68509938|ref|NP_001020271.1|Golli-mbp isoform 2 [Homo sapiens]MGNHAGKRELNAEKASTNSETNRGESEKKRNLGELSRTTSEDNEVFGEADANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDSAATSESLDVMASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIG RFFGGDRGAPKRGSGKVSSEEgi|68509930|ref|NP_001020252.1| myelinbasic protein isoform 1 [Homo sapiens]MASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKVPWLKPGRSPLPSHARSQPGLCNMYKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGT LSKIFKLGGRDSRSGSPMARRgi|4505123|ref|NP_002376.1| myelinbasic protein isoform 2 [Homo sapiens] MASQKRPSQRHGSKYLATASTM 348MDL-104238 N-[4-(4-morpholinylcarbonyl)benzoyl]-L- Anti-valyl-N′-[3,3,4,4,4-pentafluoro-1-(1- inflammatorymethylethyl)-2-oxobutyl]-L-2-azetamide 349 MDL-28050D-Glutamic acid, N-[N-[N-[N-[N-[1-[N-[1-[N- Antithrombotic[N-(3-carboxy-1-oxopropyl)-L-tyrosyl]-L- Anticoagulantalpha-glutamyl]-L-prolyl]-L-isoleucyl]-L-prolyl]-L-alpha-glutamyl]-L-alpha-glutamyl]-L-alanyl]-3-cyclohexyl-L-alanyl]- 101 MMP inhibitorsFN 439; FN-439; H2N—C6H4—CO-Gly-Pro- Antiarthritic,Leu-Ala-NHOH; MMP-inhibitor I; p-NH₂- Anticancer,Bz-Gly-Pro-D-Leu-D-Ala-NHOH Anti- inflammatory 350 MR-988N-pivaloyl-leucyl-gamma-aminobutyric acid Antipileptic 351 mertiatideGlycine, N-[N-[N- Diagnostic (mercaptoacetyl)glycyl]glycyl]- 352metkephamide L-Methioninamide, L-tyrosyl-D-alanylglycyl-Analgesic, other L-phenylalanyl-N2-methyl-, monoacetate (salt) 353murabutide D-Glutamine, N2-[N-(N-acetylmuramoyl)- Immunomodulator,L-alanyl]-, butyl ester anti-infective Anticancer, immunological Immuno-stimulant, other 354 muramyl D-alpha-Glutamine, N2-[N-(N-Immunomodulator, dipeptide acetylmuramoyl)-L-alanyl]- anti-infectivederivatives Anticancer, immunological Immunostimulant, other 355NPY24-36 N-acetyl[Leu-28Leu-31]NPY24-36 Antihypotensive 102 NAGAAsn-Ala-Gly-Ala Analgesic, other 356 tiplimotideL-Porline, D-alanyl-L-lysyl-L-prolyl-L- Multiplevalyl-L-valyl-L-histidyl-L-leucyl-L- sclerosisphenylalanyl-L-alanyl-L-asparaginyl- treatmentL-isoleucyl-L-valyl-L-threonyl-L- prolyl-L-arginyl-L-threonyl- 103opebecan gi|157276599|ref|NP_001716.2| bactericidal/ Recombinant,permeability-increasing protein precursor other [Homo sapiens]Antibacterial, MRENMARGPCNAPRWASLMVLVAIGTAVTAAVNPGVVVRISQKG otherLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMD GI inflamma-IREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMS tory/bowelGNFDLSIEGMSISADLKLGSNPTSGKPTITCSSCSSHINSVHVH disordersISKSKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSELQPY VulneraryFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENH Anti-HNPPPFAPPVMEFPAAHDRMVYLGLSDYFFNTAGLVYQEAGVLK inflammatoryMTLRDDMIPKESKFRLTTKFFGTFLPEVAKKFPNMKIQIHVSAS SymptomaticTPPHLSVQPTGLTFYPAVDVQAFAVLPNSSLASLFLIGMHTTGS antidiabeticMEVSAESNRLVGELKLDRLLLELKHSNIGPFPVELLQDIMNYIV OphthalmologicalPILVLPRVNEKLQKGFPLPTPARVQLYNVVLQPHQNFLLFGADV VYK 104 and liraglutideGLP-1 Glycine, L-histidyl-L-alanyl-L-alpha- Antidiabetic 105gluamylglycyl-L-threonyl-L-phenylalanyl-L- Anorectic/Antio-threonyl-L-seryl-L-alpha-aspartyl-L-valyl- besityL-seryl-L-seryl-L-tyrosyl-L-leucyl-L-alpha-glutamylglycyl-L-glutaminyl-L-alanyl-N6-[N-(1-oxohexadecyl)-L-gamma-glutamyl]-L-lysyl-L-alpha-glutamyl-L-phenylalanyl-L-isoleucyl-L-alanyl-L-tryptophyl-L-leucyl-L-valyl-L- arginylglycyl-L-arginyl-SFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNA NIKISGKWKAQKRFLKMSGNFDLSIE106 Nona CCK GMSISADLKLGSNPTSGKPTITCSSCSSHINSVHVHISKSKVGW DiagnosticLIQLFHKKIESALRNKMNSQVCEKVT Neuroleptic Anorectic/Antio- besityAntidepressant 107 and NP-06 Cysteinyl-leucyl-glycyl-valyl-glycyl-seryl-Antiviral, anti- 108 cysteinyl-asparaginyl-aspartyl-phenylalanyl- HIValanyl-glycyl-cysteinyl-glycyl-tyrosyl-alanyl-isoleucyl-valyl-cysteinyl- phenylalanyl-tryptophanS-3.1-S-3.13:S-3.7-S-3.19- bis(disulfide)N-2.1-C-4.9-lactamNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQ MKGEFYSENHHNPPPFAPPVMEFPAA109 NPC-18545 Bradykinin, N2-D-arginyl-3-(trans-4-hydroxy- Anti-L-proline)-7-(D-1,2,3,4-tetrahydro-3- inflammatoryisoquinolinecarboxylic acid)-3-[L- (2alpha,3aβ,7a.beta.)-octahydro-1H-indole-2-carboxylicacid]-HDRMVYLGLSDYFFNTAGLVYQEAGVLKMTLRDDMIPKESKFRL TTKFFGTFLPEVAKKFPNMKIQIHVS110 Nva-FMDP Nva-N3-4-methoxyfumaroyl-L-2,3- Antifungaldiaminopropanoic acid ASTPPHLSVQPTGLTFYPAVDVQAFAVLPNSSLASLFLIGMHTTGSMEVSAESNRLVGELKLDRLLLELK 111 nacartocin6-Carbaoxytocin, 1-(3-mercaptopropanoic Hormoneacid)-2-(4-ethyl-L-phenylalanine)- Labour inducerHSNIGPFPVELLQDIMNYIVPILVLPRVNEKLQKGFPLPTPARV Anti-QLYNVVLQPHQNFLLFGADVVYK hypertensive, diuretic 112 natural peptideU.S. Pat. No. 5,288,708 Antiulcer Partial N terminal sequence: H₂N-Gly-Hepatoprotective Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp- VulneraryAsp-Ala-Gly-Leu-Val-- . . . --COOH Anti- inflammatory AntiparkinsonianUrological  39 neriritide BNP SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRHCardiostimulant citrate Vasodilator, 113-141 neurotrophicU.S. Pat. No. 5,545,719: coronary factorsAspLeuGlnValPheVal; GlyGluLysLysAsp; CognitionAlaThrHisGluSer; CysLeuProValSerGly; enhancerLeuProValSerGlySer; ProCysHisAlaProPro;  NeuroprotectiveGlyGlyHisAspLeuGluSerGly; AspAspLeuGlnValPhe 15 ProLeuThrSerGly 15LeuIleHisPheGluGluGlyVal 15 (2) INFORMATIONFOR SEQ ID NO: 11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 11: GlyGluPheSerTyrAspSer 15 (2)INFORMATION FOR SEQ ID: 12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 12: HisAlaProProLeuThrSer 15 (2)INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 13: AspLeuGluSerGlyGluPhe 15 (2)INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 8 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 14: GlyGluPheSerValCysAspSer15 (2) INFORMATION FOR SEQ ID NO: 15: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 10amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (v) FRAGMENT TYPE: internal (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 15:LysLysGlyGluPheSerValAlaAspSer 1510 (2)INFOMATION FOR SEQ ID NO: 16: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:LysLysGlyGluPheTyrCysSerArg15 (2)INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 13 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:GlyLeuArgValArgValTrpAsnGlyLysPheProLys 1510(2) INFORMATION FOR SEQ ID NO: 18: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 16amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:GlyValAlaPheGluGluAlaProAspAspHisSerPhePheLeuPhe 151015 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (v) FRAGMENT TYPE: internal (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 19: GlyGlyHisAspLeuSerGly 15 (2)INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 8 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 20: GlyGlyHisAspLeuGluSerGly 15(2) INFORMATION FOR SEQ ID NO: 21: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 14amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (v) FRAGMENT TYPE: internal (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 21:GlyGlyHisAspLeuGluSerGlyGluPheSerTyrAspSer1510 (2) INFORMATION FOR SEQ ID NO: 22: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 14amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (v) FRAGMENT TYPE: internal (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 22:GlyGlySerAspLeuSerGlyGluPheSerValCysAspSer1510 (2) INFORMATION FOR SEQ ID NO: 23: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 15amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (v) FRAGMENT TYPE: internal (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 23: GlyGlySerAspLeuSerGlyGlyGluPheSerValCysAspSer 151015 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:15 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (v) FRAGMENT TYPE: internal (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 24:GlyGlySerAspLeuSerGlyGlyGluPheSerValAlaAspSer 151015 (2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH:14 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (v) FRAGMENT TYPE: internal (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 25:GlyGlySerAspLeuSerGlyGluPheSerValAlaAspSer1510 (2) INFORMATION FOR SEQ ID NO: 26:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH:6 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:GluThrLeuGlnPheArg 15 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 8 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:LysLysGluThrLeuGlnPheArg 15 (2)INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 8 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION:SEQ ID NO:28: GluThrLeuGlnPheArgLysLys 15(2)INFORMATION FOR SEQ ID NO: 29: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 9amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE  DESCRIPTION: SEQ ID NO: 29:LysAlaSerThrThrThrAsnTyrThr 15 357 nifalatideL-Prolinamide, L-tyrosyl-4-(methylsulfinyl)- AntidiarrhoealD-2-aminobutanoylglycyl-4-nitro-L- Analgesic, other phenylalanyl- 358Org-2766 L-Phenylalanine, 4-(methylsulfonyl)-L- ACTH2-aminobutanoyl-L-alpha-glutamyl-L- Symptomatichistidyl-L-phenylalanyl-D-lysyl- antidiabetic Radio/chemo- protectiveNeurological 359 Org-30035 L-Phenylalanine, glycylglycyl-L- Neurolepticphenylalanyl-4-(methylsulfonyl)-L-2- Anxiolytic aminobutanoyl-D-lysyl-360 octreotide somato- L-Cysteinamide, D-phenylalanyl-L-cysteinyl-Acromegaly statin L-phenylalanyl-D-tryptophyl-L-lysyl-L- Antidiarrhoealthreonyl-N-[2-hydroxy-1- Anticancer,(hydroxymethyl)propyl]-, cyclic (2-7)- hormonaldisulfide [R-(R*,R*)]-; L-Cysteinamide,D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-(2- hydroxy-1-(hydroxymethyl)propyl)-,cyclic (2-7)-disulfide, (R-(R*,R*))- 142 osteogenicGlycine, L-alanyl-L-leucyl-L-lysyl-L- Osteoporosis growth peptidearginyl-L-glutaminylglycyl-L-arginyl- treatmentL-threonyl-L-leucyl-L-tyrosylglycyl-L- phenylalanylglycyl- 143 P-113Angiotensin II, 1-(N-methylglycine)-5-L- Stomatologicalvaline-8-L-alanine-[CAS]; (Sar(1), Ala (8)) Antibacterial,ANGII; (Sar1, Val5, Ala8) Angiotensin II; other1 Sar 8 Ala Angiotensin II; 1 Sarcosine 8 AntifungalAlanine Angiotensin II; 1-Sar-8-AlaAngiotensin II; 1-Sar-8-Ala-angiotensin II;1-Sarcosine-8-Alanine Angiotensin II;Acetate, Hydrated Saralasin; Angiotensin II,1-Sar-8-Ala; Angiotensin II, 1-Sarcosine-8-Alanine; Anhydrous Saralasin Acetate;Hydrated Saralasin Acetate; P-113; P-113Acetate; Sar Arg Val Tyr Val His Pro Ala;Sar-Arg-Val-Tyr-Val-His-Pro-Ala; SaralasinAcetate; Saralasin Acetate, Anhydrous;Saralasin Acetate, Hydrated; angiotensin II,Sar(1)-Ala(8)-; angiotensin II, sarcosyl (1)-alanine(8)- 361 PACAP 27Pituitary adenylate cyclase-activating Antiviral, anti- peptide-27 HIV362 PAPP N-(dibenzyloxyphosphophionyl)-L-alanyl- Anti-L-prolyl-L-proline hypertensive, other 363 PD-83176CBZ-his-tyr(OBn)-ser(OBn)-trp-D-ala-NH₂ Anticancer, other 364 PD-122264N-[(1,1-dimethylethoxy)carbonyl]-alpha- Anorectic/Antio-methyltryptophyl-L-phenylalaninamide besity Analgesic, other 365PD-132002 DL-Serinamide, N-(4-morpholinylsulfonyl)- Anti-L-phenylalanyl-N-[1-(cyclohexylmethyl)- hypertensive,2,3-dihydroxy-5-methylhexyl]-O-methyl-3- renin systemoxo-, [1S-(1R*,2S*,3R*)]- 144 PenetratinU.S. Pat. Nos. 5,888,762 and 6,080,762; PCT FormulationPub. Nos. WO/2000/29427 and WO/2000/01417: technologyNH2-Arg Lys Arg Gly Arg Gln Thr Tyr Thr ArgTyr Gln Thr Leu Glu Leu Glu Lys Glu Phe HisPhe Asn Arg Tyr Leu Thr Arg Arg Arg Arg IleGlu Ile Ala His Ala Leu Cys Leu Thr Glu ArgGln Ile Lys Ile Trp Phe Gln Asn Arg Arg MetLys Trp Lys Lys Glu Asn-COOH. 366 PL-030Glycinamide, L-tyrosyl-L-prolyl-N- Analgesic, othermethyl-L-phenylalanyl-D-prolyl- 367 POL-443 Z-prolyl-leucyl-tryptophanAnti- hypertensive, renin system 368 POL-509L-Tryptophan, N-[N-(5-oxo-L-prolyl)-L- Immunostimulant,leucyl]-, methyl ester- other 369 PPA D-phenylalanine-L-proline-L-Anticoagulant arginylchloromethane Diagnostic Antithrombotic 145 PR-39L-Prolinamide, L-arginyl-L-arginyl-L- Antibacterial,arginyl-L-prolyl-L-arginyl-L-prolyl-L- otherprolyl-L-tyrosyl-L-leucyl-L-prolyl-L-arginyl-L-prolyl-L-arginyl-L-prolyl-L- prolyl-L-prolyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-prolyl-L-arginyl-L-leucyl-L-prolyl-L-prolyl-L-arginyl-L-isoleucyl-L-prolyl-L-prolylglycyl-L-phenylalanyl-L-prolyl-L-prolyl-L-arginyl-L-phenylalanyl-L-prolyl-L-prolyl-L- arginyl-L-phenylalanyl- 146tigapotide L-Threonine, L-alpha-glutamyl-L-tryptophyl- Anticancer,triflutate L-glutaminyl-L-threonyl-L-alpha-aspartyl- otherL-asparaginyl-S-[(acetylamino)methyl]-L-cysteinyl-L-glutamyl-L-threonyl-S- [(acetylamino)methyl]-L-cysteinyl-L-threonyl-S-[(acetylamino)methyl]-L-cysteinyl-L-tyrosyl-L-alpha-glutamyl-, mono(trifluoroacetate) 370 PT-14L-Lysinamide, N-acetyl-L-norleucyl-L-alpha- Male sexualaspartyl-L-histidyl-D-phenylalanyl-L- dysfunctionarginyl-L-tryptophyl-, cyclic (2-7)-peptide Female sexual dysfunction147 PT-5 somato- gi|21619156|gb|AAH32625.1| Somatostatin Anticancer,statin [Homo sapiens] other MLSCRLQCALAALSIVLALGCVTGAPSDPRLRQFLQKSLAAAAGKQELAKYFLAELLSEPNQTENDALEPEDLSQAAEQDEMRLELQRSANSNPAMAPRERKAGCKNFFWKTFTSC 148 semparatide PTHrPgi|131542|sp|P12272.1|PTHR_HUMAN HormoneParathyroid hormone-related protein Osteoporosisprecursor (PTH-rP) (PTHrP) [Contains: treatmentPTHrP[1-36]; PTHrP[38-94]; Osteostatin (PTHrP[107-139])]MQRRLVQQWSVAVFLLSYAVPSCGRSVEGLSRRLKRAVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVFGSDDEGRYLTQETHKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSRRH 149 parathyroid PTHSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF Osteoporosis hormone treatmentfragments 150 enfuvirtide L-Phenylalaninamide, N-acetyl-L-tyrosyl-L-Antiviral, anti- threonyl-L-seryl-L-leucyl-L-isoleucyl-L- HIVhistidyl-L-seryl-L-leucyl-L-isoleucyl-L-alpha-glutamyl-L-alpha-glutamyl-L-seryl-L-glutaminyl-L-asparaginyl-L-glutaminyl-L-glutaminyl-L-alpha-glutamyl-L-lysyl-L-asparaginyl-L-alpha-glutamyl-L-glutaminyl-L-alpha-glutamyl-L-leucyl-L-leucyl-L-alpha-glutamyl-L-leucyl-L-alpha-aspartyl-L-lysyl-L-tryptophyl-L-alanyl-L-seryl-L-leucyl-L-tryptophyl-L-asparaginyl-L-tryptophyl- 151 pentapeptideAla-Arg-Pro-Ala-Lys Vasodilator, 6A coronary 371 pentigetideL-Arginine, N2-[1-[N-(N-L-alpha-aspartyl-L- Antiallergic,seryl)-L-alpha-aspartyl]-L-prolyl]- non-asthma OphthalmologicalAntiasthma 372 peptide N1,N3-bis(2,3-dihydroxypropyl)-2,4,6-Ophthalmological analogues triiodo-5-(2-methoxyacetamido)-N1-Antiarthritic, methylisophthalamide other Antiulcer Anti- hypertensive,other Multiple sclerosis treatment COPD treatment 373 peptide G[Arg(6),D-Trp(7,9),MePhe(8)]substance P Anticancer, other 374 peptide TD-Ala1-peptide T Antiviral, anti- analogue HIV 375 peptide TL-Threonine, N-[N-[N2-[N-[N-[N-(N-L- Analgesic, otheralanyl-L-seryl)-L-threonyl]-L- Antiviral, otherthreonyl]-L-threonyl]-L-asparaginyl]-L- Antiarthritic, tyrosyl]- otherGI inflamma- tory/bowel disorders Anti- inflammatory 152 pramlintide1,2-Dithia-5,8,11,14,17- Antidiabeticpentaazacycloeicosane, cyclic peptide Anorectic/Antio- derivative besityU.S. Pat. No. 5,998,367 gi|10066209|gb|AAE39671.1| Sequence 1from U.S. Pat. No. 5,998,367 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY; 376pranlukast Benzamide, N-[4-oxo-2-(1H-tetrazol-5-yl)-4H- Antiasthma1-benzopyran-8-yl]-4-(4-phenylbutoxy)-; 8- Antiallergic,(4 (4-phenylbutoxy)benzoyl)amino-2- non-asthma(tetrazol-5′-yl)-4-oxo-4H-1-benzopyran   3 proinsulin proinsu-gi|59036749|gb|AAW83741.1| proinsulin Antidiabetic lin [Homo sapiens]MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQK RGIVEQCCTSICSLYQLENYCN 377protirelin TRH L-Prolinamide, 5-oxo-L-prolyl-L-histidyl-; Releasing2-Nle-3-Prot-protirelin; TRH, Nle(2)- hormoneProt(3)-; pyroglutamyl-norleucyl-proline Diagnostic thioamide 378protierlin TRH prolinamide, 5-oxo-L-prolyl-L-histidyl- Releasing hormoneCognition enhancer 153 Ro-25-1553L-Threoninamide, N-acetyl-L-histidyl-L- Antiasthmaseryl-L-alpha-aspartyl-L-alanyl-L-valyl-L- Anti-phenylalanyl-L-threonyl-L-alpha-glutamyl- inflammatoryL-asparaginyl-L-tyrosyl-L-threonyl-L-lysyl-L-leucyl-L-arginyl-L-lysyl-L-glutaminyl-L-norleucyl-L-alanyl-L-alanyl-L-lysyl-L-lysyl-L-tyrosyl-L-leucyl-L-asparaginyl-L-alpha-aspartyl-L-leucyl-L-lysyl-L-lysylglycylglycyl-, (25-21)-lactam 379 RWJ-51438N-methylphenylalanyl-N-(4- Antithrombotic((aminoiminomethyl)amino)-1-((6-carboxy-2-benzothiazolyl)carbonyl)butyl)prolinamide 380 TRH TRHL-Prolinamide, 5-oxo-L-prolyl-L-histidyl- Diagnostic3,3-dimethyl-; pyroGlu-His-Pro-NH₂ (or Thyroid hormone5-oxo-L-prolyl-L-histidyl-L-prolinamide Releasing hormone 154 reninBoc-Leu-Lys-Arg-Met-Pro-OMe Anti- inhibitors hypertensive, 381 romurtideL-Lysine, N2-[N2-[N-(N-acetylmuramoyl)-L- Radio/chemo-alanyl]-D-alpha-glutaminyl]-N6-(1- protectiveoxooctadecyl)-; L-Lysine, N2-(N2-(N-(N- Immunostimulant,acetylmuramoyl)-L-alanyl)-D-alpha- otherglutaminyl)-N6-(1-oxooctadecyl)- 382 S-17162 L-Tryptophan, N-[(2,3-Urological dihydroxypropoxy)hydroxyphosphinyl]-L- leucyl-, disodium salt383 S-2441 L-Argininamide, D-prolyl-L- Antimigrainephenylalanyl-N-heptyl- Antigout Septic shock treatment 384 SDZ-CO-611somato- L-Cysteinamide, N-(1-deoxy-4-O-.alpha.-D- Somatostatin statinglucopyranosyl-D-fructopyranos-1-yl)-D-phenylalanyl-L-cystinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-, cyclic (2.fwdarw.7)-disulfide, [R-(R*,R*)]-385 SK & F-101926 L-Argininamide, O-ethyl-N-[(1- Anti-mercaptocyclohexyl)acetyl]-D-tyrosyl-L- hypertensive,phenylalanyl-L-valyl-L-asparaginyl-L- diureticcysteinyl-L-prolyl-, cyclic (1-5)-disulfide 386 SK & F-110679His-D-Trp-Ala-Trp-D-Phe-LysNH₂ Releasing hormone Vulnerary 387edotreotide [N-[2-[4,7-Bis[(carboxy-kappaO)methyl]-10- Anticancer,(carboxymethyl)-1,4,7,10-tetraazacyclododec- hormonal1-yl-kappaN1,kappaN4,kappaN10]acetyl]-D-phenylalanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-threonyl-L-cysteinyl- L-threoninol cyclic (2-7)-disulfidato(3-)]yttrium 155 SP-1 pGlu-Glu-Asp-Cys-Lys Anticancer, other156 SPAAT L-Lysine, L-methionyl-L-phenylalanyl-L- COPD treatmentleucyl-L-alpha-glutamyl-L-alanyl-L-isoleucyl-L-prolyl-L-methionyl-L-seryl-L-isoleucyl-L-prolyl-L-prolyl-L-alpha-glutamyl-L-valyl-L-lysyl-L-phenylalanyl-L-asparaginyl-L-lysyl-L-prolyl-L- phenylalanyl-L-valyl-L-phenylalanyl-L-leucyl-L-methionyl-L-isoleucyl-L-alpha-glutamyl-L-glutaminyl-L-asparaginyl-L-threonyl-L-lysyl-L-seryl-L-prolyl-L-leucyl-L-phenylalanyl-L-methionylglycyl-L-lysyl-valyl-L-valyl-L-asparaginyl-L-prolyl-L- threonyl-L-glutaminyl- 388SR-41476 Z-Tyr-Val-Sta-Ala-Sta-OMe Antiviral, anti- HIV 389 SR-421281-[N-(3-methyl-1-oxobutyl)-L- Anti- phenylalanine]-2-L-norleucine-hypertensive, renin system 157 SR-42654isoval-phe-norleu-sta-ala-sta-lys Anti- hypertensive, renin system 147SRIF-A somato- gi|21619156|gb|AAH32625.1| Somatostatin Somatostatinstatin [Homo sapiens] HaemostaticMLSCRLQCALAALSIVLALGCVTGAPSDPRLRQFLQKSLAAAAG Alimentary/Meta-KQELAKYFLAELLSEPNQTENDALEPEDLSQAAEQDEMRLELQR bolic, otherSANSNPAMAPRERKAGCKNFFWKTFTSC 8-D-tryptophan-14-D-cysteinesomatostatin(sheep) 158 calcitonin calcito- CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTPOsteoporosis nin treatment 390 salmon calcito-11,18-Arg-14-Lys-salmon calcitonin; Osteoporosis calcitonin nin11,18-arginyl-14-lysine-salmon calcitonin; treatmentArg-Lys-Arg-CT; calcitonin, salmon, arginyl(11,18)-lysine(14)- 159sermorelin Tyr-Ala-Asp-Ala-Ile-Phe-Asn-Ser-Tyr-Arg-Lys- IdiopathicVal-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu- growth hormoneGln-Asp-Ile-Met-Ser-Arg-NH₂ deficiency Imaging agent 391 saralasin1-Sar-8-Ala-angiotensin; Angiotensin II, Anti- acetate1-(N-methylglycine)-5-L-valine-8-L-alanine- hypertensive, renin system160 secretin His-Ser-Asp-Gly-Thr-Phe-OMe; histidyl- Haemostatic;seryl-aspartyl-glycyl-threonyl- pancreatic phenylalanine-O-methyl-dysfunction (diagnostic), asthma, COPD, others 159 sermorelinTyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg- Releasing acetateLys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu- hormoneLeu-Gln-Asp-Ile-Met-Ser-Arg-NH₂ Diagnostic 159 sermorelinTyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH₂ 161 sinapultideL-Lysine, L-lysyl-L-leucyl-L-leucyl-L- Lung Surfactantleucyl-L-leucyl-L-lysyl-L-leucyl-L-leucyl-L-leucyl-L-leucyl-L-lysyl-L-leucyl-L-leucyl-L-leucyl-L-leucyl-L-lysyl-L-leucyl- L-leucyl-L-leucyl-L-leucyl-162 sleep inducing Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu Hypnotic/Seda-peptide tive Dependence treatment 163 somatoliberingi|11034841|ref|NP_066567.1| growth hormone Growth hormonereleasing hormone preproprotein Releasing [Homo sapiens] hormoneMPLWVFFFVILTLSNSSHCSPPPPLTLRMRRYADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARLGRQVDSMWAEQKQ MELESILVALLQKHSRNSQG 164PTR-3173 somato- Cyclic[(R)-βMeNphe-Phe-DTrp-Lys-Thr-Phe], Acoegalystatin MPLWVFFFVILTLSNSSHCSPPPPLTLRMRRYADAIFTNSYRKV SymptomaticLGQLSARKLLQDIMSRQQGESNQERG antidiabetic Ophthalmological UrologicalAnticancer, hormonal 165 somatostatin somato-des-(Ala1,Gly2)-(D-Trp8,D-Trp8,D-Asu(3,14))- Acromegaly analogue statinsomatostatin, Antidiabetic ARARLGRQVDSMWAEQKQMELESILVALLQKHSRNSQGDiagnostic 392 somatostatin somato-cyclo-(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe)- Acromegaly analogues statinsomatostatin Antidiabetic 393 somatostatin somato-3,14-Dicarbasomatostatin, 1-de-L-alanine-2- Acromegaly statindeglycine-3-butanoic acid-11-L-tyrosine- 394 somatostatin somato-3,14-Dicarbasomatostatin, 1-de-L-alanine- Acromegaly statin2-deglycine-3-butanoic acid-11-L-tyrosine- 395 syndyphalinGlycinamide, L-tyrosyl-4-(methylsulfinyl)- Analgesic, otherD-2-aminobutanoyl-N-methyl-N-(2- phenylethyl)- 166 syntheticgi|109948285|ref|NP_001035971.1| poly(A) Antiulcer peptide BPCbinding protein, cytoplasmic 1-like 2B Hepatoprotective [Homo sapiens]Vulnerary MASLYVGDLHPEVTEAMLYEKFSPAGPILSIRICRDKITRRSLG Anti-YAYVNYQQPVDAKRALETLNFDVIKGRPVRIMWSQRDPSLRKSG inflammatoryVGNVFIKNLGKTIDNKALYNIFSAFGNILSCKVACDEKGPKGYG AntiparkinsonianFVHFQKQESAERAIDVMNGMFLNYRKIFVGRFKSHKEREAERGA MusculoskeletalWARQSTSADVKDFEEDTDEEATLR 167 T22 L-Argininamide, L-arginyl-L-arginyl-L-Antiviral, anti- tryptophyl-L-cysteinyl-L-tyrosyl-L-arginyl- HIVL-lysyl-L-cysteinyl-L-tyrosyl-L-lysylglycyl-L-tyrosyl-L-cysteinyl-L-tyrosyl-L-arginyl-L-lysyl-L-cysteinyl-, cyclic (4-17),(8-13)-bis(disulfide) 396Tc-99m Technetium-99Tc, (cyclo(L-homocysteinyl- Imaging agent depreotideN-methyl-L-phenylalanyl-L-tyrosyl-D- tryptophyl-L-lysyl-L-valyl)(1-1′)-thioether with 3- ((mercaptoacetyl)amino)-L-alanyl-L-lysyl-L-cysteinyl-L-lysinamidato(3-)) oxo-, (SP-5-24)- 397 Tc-99m-P28013, 13′-[Oxybis[methylene(2,5-dioxo-1,3- Imaging agentpyrrolidinediyl)]]bis[N-(mercaptoacetyl)- AntithromboticD-tyrosyl-S-(3-aminopropyl)-L- cysteinylglycyl-L-alpha-aspartyl-L-cysteinylglycylglycyl-S- [(acetylamino)methyl]-L-cysteinylglycyl-S-[(acetylamino)methyl-L-cysteinylglycylglycyl-L-cysteinamide], cyclic (1 → 5), (1′ →5′), -bis(sulfide) 398 TEI-1345(7E)-8-(2-naphthyl)-5,6-trans-5,6-methano-7- Anti-octenyl 3-(3,4-dimethoxyphenyl)-2-propenoate inflammatory 168 THFLeu-Glu-Asp-Gly-Pro-Lys-Phe-Leu; leucyl- Immunomodulator,glutamyl-aspartyl-glycyl-proly-lysyl- anti-infective,phenylalanyl-leucine Immunostimulant, anti-AIDS 169 Theradigm-Dipalmitoyl-Lys-Ser-Ser-Gln-Tyr-Ile- Immunomodulator, HBVLys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile- anti-infectiveThr-Glu-Ala-Ala-Ala-Phe-Leu-Pro-Ser- ImmunostimulantAsp-Phe-Phe-Pro-Ser-Val-OH  80 tesamorelin GHRF gi|337133|gb|AAA52609.1|growth hormone Musculoskeletal, acetate releasing factor COPD,MPLWVFFFVILTLSNSSHCSPPPPLTLRMRRYADAIFT Hypnotic/Seda-NSYRKVLGQLSARKLLQDIMSRQQGESNQERGARAR tive,LGRQVDSMWAEQKQMELESILVALLQKHRNSQG Immunostimulant,(3E)-Hex-3-enoylsomatoliberin (human) Antidiabetic, acetate (salt)Anabolic, Symptomatic antidiabetic, Vulnerary 170 TP-9201L-Cysteinamide, N-acetyl-L-cysteinyl-L- Neuroprotective,asparaginyl-L-prolyl-L-arginylglycyl-L- Antithrombotic,alpha-aspartyl-O-methyl-L-tyrosyl-L- Antianginal,arginyl-, cyclic (1-9)-disulfide Cardiovascular 399 TRH analogues TRHpyroGlu-His-Pro-NH₂ (or 5-oxo-L-prolyl- CognitionL-histidyl-L-prolinamide) enhancer 400 TT-235[β,β-(3-Thiapentamethylene)-β- Labour inhibitorsulfanylpropionic acid, D- Trp2,Pen6,Arg8]-oxytocin acetate 401tabilautide L-Lysinamide, 6-carboxy-N6-[N-[N-(1- Immunomodulator,oxododecyl)-L-alanyl]-D-gamma- anti-infective glutamyl]-, (S)-Radio/chemo- protective Immunostimulant, other 171 and terlipressinN-[N-(N-glycylglycyl)glycyl]-8-lysine-; Haemostatic; GI 172Gly-Gly-Gly-8-Lys-vasopressin; N-(alpha)- bleedingglycyl-glycyl-glycyl-8-lysine vasopressin;Gly-Gly-Gly-c[Cys-Tyr-Phe-Gln-Asn-Cys]-Pro-Lys-Gly-NH₂; N-(N-(N-glycylglycyl)glycyl)- 8-L-lysinevasopressin 171 andterlipressin N-[N-(N-glycylglycyl)glycyl]-8-lysine-; Haemostatic; GI 172Gly-Gly-Gly-8-Lys-vasopressin; N-(alpha)- bleedingglycyl-glycyl-glycyl-8-lysine vasopressin;Gly-Gly-Gly-c[Cys-Tyr-Phe-Gln-Asn-Cys]-Pro- Lys-Gly-NH₂ 402 teverelixD-Alaninamide, N-acetyl-3-(2- Anticancer,naphthalenyl)-D-alanyl-4-chloro-D- hormonalphenylalanyl-3-(3-pyridinyl)-D-alanyl- ProstateL-seryl-L-tyrosyl-N6-(aminocarbonyl)- disordersD-lysyl-L-leucyl-N6-(1-methylethyl)-L- Menstruation lysyl-L-prolyl-disorders Fertility enhancer Male contraceptive 403 thymopentinL-Tyrosine, N-[N-[N-(N2-L-arginyl-L- Immunostimulant,lysyl)-L-alpha-aspartyl]-L-valyl]-; L- otherTyrosine, N-(N-(N-(N2-L-arginyl-L- Immunomodulator,lysyl)-L-alpha-aspartyl)-L-valyl)- anti-infective 404 triletideL-Histidine, N-[N-(N-acetyl-L-phenylalanyl)- AntiulcerL-phenylalanyl]-, methylester 405 tuftsinL-Arginine, N2-[1-(N2-L-threonyl-L- Anticancer, lysyl)-L-prolyl]-immunological Immunostimulant, other 173 UroguanylinGuanylin (rat reduced), 1-L-glutamine-2-L- Alimentary/Meta-glutamic acid-3-L-aspartic acid-6-L-leucine- bolic, other8-L-isoleucine-9-L-asparagine-10-L-valine- Antidiarrhoeal Diagnostic 174VIC gi|6681267|ref|NP_031929.1| endothelin Gastroprokinetic3 [Mus musculus] MEPGLWLLLGLTVTSAAGLVPCPQSGDSGRASVSQGPPEAGSERGCEETVAGPGERIVSPTVALPAQPESAGQERAPGRSGKQEDKGLPAHHRPRRCTCFTYKDKECVYYCHLDIIWINTPEQTVPYGLSNYRESLRGKRSLGPVPESSQPSPWTRLRCTCMGADDKACAHFCARTRDVTSYSGRAERPAAEEMRETGGPRQRLMSRTDKAHRP 175 VIP derivativegi|5803023|ref|NP_006807.1| lectin, Antiasthmamannose-binding 2 [Homo sapiens] Vasodilator,MAAEGWIWRWGWGRRCLGRPGLLGPGPGPTTPLFLLLLLGSVTA peripheralDITDGNSEHLKREHSLIKPYQGVGSSSMPLWDFQGSTMLTSQYVRLTPDERSKEGSIWNHQPCFLKDWEMHVHFKVHGTGKKNLHGDGIALWYTRDRLVPGPVFGSKDNFHGLAIFLDTYPNDETTERVFPYISVMVNNGSLSYDHSKDGRWTELAGCTADFRNRDHDTFLAVRYSRGRLTVMTDLEDKNEWKNCIDITGVRLPTGYYFGASAGTGDLSDNHDIISMKLFQLMVEHTPDEESIDWTKIEPSVNFLKSPKDNVDDPTGNFRSGPLTGWRVFLLLLCALLGIVVCAVVGAVVFQKRQERN KRFY 147 vapreotide,somato- gi|21619156|gb|AAH32625.1| Somatostatin Formulation,  immediate-statin [Homo sapiens] modified- releaseMLSCRLQCALAALSIVLALGCVTGAPSDPRLRQFLQKSLAAAAG release,KQELAKYFLAELLSEPNQTENDALEPEDLSQAAEQDEMRLELQR immediateSANSNPAMAPRERKAGCKNFFWKTFTSC SomatostatinL-Tryptophanamide, D-phenylalanyl-L- Haemostaticcysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L- Anticancer,valyl-L-cysteinyl-, cyclic (2-7)-disulfide- hormonal AntidiarrhoealGI inflamma- tory/bowel disorders 406 PharmaprojectsL-Proline, 1-[N-[N-[1-[4-(4-hydroxyphenyl)- Vasodilator, No. 12691-oxobutyl]-L-prolyl]-.alpha.-methyl-DL- renal phenylalanyl]glycyl]- 407Pharmaprojects N(α)-((3S)-1-oxo-1,2,3,4- Neuroleptic No. 1583tetrahydroisoquinoline-3-carbonyl)-L- Antiparkinsonianhistidyl-L-prolinamide 408 PharmaprojectsD-2-phenylglycyl-D-2-phenylglycine Anticancer, No. 1626 immunologicalImmunostimulant, other 409 Pharmaprojects N-acyl-D-glutamyl-1-meso-Immunomodulator, No. 1779 diaminopimelyl-l-lysine tripeptideanti-infective derivatives Immunostimulant, other 176 PharmaprojectsThr-Asp-Ser-Phe-Val-Gly-Leu- Anti- No. 1876 Methionylamide hypertensive,other 410 Pharmaprojects L-leucyl-D-methionyl-glucyl-N-(2- Anti-No. 1913 adamantyl)-L-phenylalanylamide hypertensive, renin system 177Pharmaprojects Lys-Pro-Gly-Glu-Pro-Gly-Pro-Lys Anticoagulant No. 1939 178-182, Pharmaprojects U.S. Pat. No. 4,461,724 and European Antiulcer 178, No. 2063 Patent No. EP0078228: GSHK; ASHK; A_(D)SHK;Antithrombotic 183-185 LSHK; TSHK; YSHK; GSHKCH₃COOH•H₂O; SAR-SHK;and 178 PSHK; (PYR)ESHK; WSHK; GSHK•2TosOH 411 PharmaprojectsN-methyl-D-Phe-Pro-Arg-H Antithrombotic No. 2363 186 PharmaprojectsN-3-(4-hydroxyphenyl)propionyl-Pro-Hyp- Antiarrhythmic No. 2388Gly-Ala-Gly 412 Pharmaprojects Glp-lys-NH₂-L-mandelate Anticancer,No. 2425 immunological Immunostimulant, other 413 PharmaprojectsD-1-Tiq-Pro-Arg-H-sulfate Antithrombotic No. 3341 414 Pharmaprojects(2R,4S,5S,1′S)-5-(t-butoxycarbonyl)amino- Antiviral, anti- No. 34154-hydroxy-N-[1′-isopropyl-1′-(4- HIVisopropylcarbonylimidazol-2-yl)]methyl-6-phenyl-2-phenylmethyl-hexanamide 415 PharmaprojectsPiv-1-Ser-Leu-GABA, and Piv-Ser-Leu-GABA Neurological No. 4004 416Pharmaprojects (1R,4aR,8aR)-1,2,3,4,5,6,7,8- Antithrombotic No. 4323perhydroisoquinolin-1-carbonyl-(L)- Anticoagulantprolinyl-(L)-arinine aldehyde 187, and PharmaprojectsH-Trp-Ala-Ser-Gly-L-Asn-OH & H-Trp-D- Hypnotic/Seda- 417 No. 491Ala-Ser-Gly-Asp(OH)₂ Neuroprotective tive Antidepressant Neuroprotective188 Pharmaprojects H₂N-Asp-Ala-Asp-Pro-Arg-Gln-Tyr-Ala-COOH Anti-No. 4975 inflammatory 418 Pharmaprojects2-Amino-N-{1-(R)-benzyloxymethyl-2-[4- Osteoporosis No. 5200(morpholine-4-carbonyl)-4-phenyl-piperidin- treatment1-yl]-2-oxo-ethyl}-isobutyramide 419 Pharmaprojects4-chloro-phenylcarbamoyl-thienylalanyl- Anti- No. 5356leucyl-phenylalanine inflammatory Anti-infective, other 420 DMP-444synthetic cyclic pentapeptide (cyclo(D-Val- Imaging agentNMeArg-Gly-Asp-Mamb)) with a tetheredhydrazinonicotinyl (HYNIC) chelator for rediolabelling with 99mTc 189RIP-3 MSCVKLWPSGAPAPLVSIEELENQELVGKGGFGTVFRAQHRKWG Anticancer,YDVAVKIVNSKAISREVKAMASLDNEFVLRLEGVIEKVNWDQDP otherPKPALVTKFMENGSLSGLLQSQCPRPWPLLCRLLKEVVLGMFYLHDQNPVLLHRDLKPSNVLLDPELHVKLADFGLSTFQGGSQSGTGSGEPGGTLGYLAPELFVNVNRKASTASDVYSFGILMWAVLAGREVELPTEPSLVYEAVCNRQNRPSLAELPQAGPETPGLEGLKELMQLCWSSEPKDRPSFQECLPKTDEVFQMVENNMNAAVSTVKDFLSQLRSSNRRFSIPESGQGGTEMDGFRRTIENQHSRNDVMVSEWLNKLNLEEPPSSVPKKCPSLTKRSRAQEEQVPQAWTAGTSSDSMAQPPQTPETSTFRNQMPSPTSTGTPSPGPRGNQGAERQGMNWSCRTPEPNPVTGRPLVNIYNCSGVQVGDNNYLTMQQTTALPTWGLAPSGKGRGLQHPPPVGSQEGPKDPEAWSRPQGWYNHSGK 421 PharmaprojectsN-(N-acetyl-l-isoleucyl-L-tyrosyl)-(−)- Anti- No. 955 1-amino-2-(4-hypertensive, hydroxyphenyl)ethylphosphonic acid other 422 leuprolide6-D-leucine-9-(N-ethyl-L-prolinamide)- Formulation, 10-deglycinamide-modified release, Anticancer 190 edratideL-glycyl-L-tyrosyl-L-tyrosyl-L-tryptophyl- Immuno-L-seryl-L-tryptophyl-L-isoleucyl-L-arginyl- suppressantL-glutaminyl-Lprolyl-L-prolyl-L-glycyl-L-lysyl-L-glycyl-L-glutamyl-L-glutamyl-L- tryptophyl-L-isoleucyl-L-glycine423 Prosaptide H-Thr-D-Ala-Leu-Ile-Asp-Asn-Asn-Ala- Symptomatic TX14(A)Thr-Glu-Glu-Ile-Leu-Tyr-OH antidiabetic Neurological Analgesic, other  8 GLP-1 GLP-1 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Antidiabetic 160secretin His-Ser-Asp-Gly-Thr-Phe-OMe; histidyl- Hormone,seryl-aspartyl-glycyl-threonyl- Diagnostic, GI phenylalanine-O-methyl-inflamma- tory/bowel disorders, Neurological, Neuroleptic 147 BIM-23190somato- gi|21619156|gb|AAH32625.1| Somatostatin Acromegaly statin[Homo sapiens] Antidiabetic MLSCRLQCALAALSIVLALGCVTGAPSDPRLRQFLQKSLAAAAGKQELAKYFLAELLSEPNQTENDALEPEDLSQAAEQDEMRLELQRSANSNPAMAPRERKAGCKNFFWKTFTSC L-Threoninamide, N-[[4-(2-hydroxyethyl)-1-piperazinyl]acetyl]-D-phenylalanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-(2S)-2-aminobutanoyl-L-cysteinyl-, cyclic (2-7)-disulfide 424leuprorelin 6-D-leucine-9-(N-ethyl-L-prolinamide)- Formulation,10-deglycinamide- Anticancer 191 β-amyloid beta-gi|8176533|gb|AAB26264.2| beta-amyloid Cognition peptides amyloidpeptide precursor; beta APP [Homo sapiens] enhancer peptideGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIIITLVMLKKQYTSNHHGVVE 425 oglufanideL-tryptophan, L-alpha-glutamyl-, Immunomodulator, disodium disodium saltanti-infective Anticancer, immunological 192 HAV peptideleucyl-arginyl-alanyl-histidyl-alanyl-valyl- Neurological matrixaspartyl-valyl-asparaginyl-glycinamide 149 PTH 1-34 PTHSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF Hormone leuprorelin6-D-leucine-9-(N-ethyl-L-prolinamide)- Anticancer 10-deglycinamide- 193TRP-2 H-Leu-Leu-Pro-Gly-Gly-Arg-Pro-Tyr-Arg-OH Anticancer, immunological426 golotimod (2R)-2-amino-5-[[(1S)-1-carboxy-2-(1H-indol-Immunostimulant, 3-yl)ethyl]amino]-5-oxopentanoic acid otherImmunomodulator, anti-infective Anticancer, immunological Stomatological194 angiotensin-II Angioten- gi|28710|emb|CAA77513.1| angiotensin IIVulnerary sin II [Homo sapiens] SymptomaticMILNSSTEDGIKRIQDDCPKAGRHNYIFVMIPTLYSIIFVVGIF antidiabeticGNSLVVIVIYFYMKLKTVASVFLLNLALADLCFLLTLPLWAVYTAMEYRWPFGNYLCKIASASVSFNLYASVFLLTCLSIDRYLAIVHPMKSRLRRTMLVAKVTCIIIWLLAGLASLPAIIHRNVFFIENTNITVCAFHYESQNSTLPIGLGLTKNILGFLFPFLIILTSYTLIWKALKKAYEIQKNKPRNDDIFKIIMAIVLFFFFSWIPHQIFTFLDVLIQLGIIRDCRIADIVDTAMPITICIAYFNNCLNPLFYGFLGKKFKRYFLQLLKYIPPKAKSHSNLSTKMSTLSYRPSDNVSSSTKKP APCFEVE 195 omigananL-lysinamide, L-isoleucyl-L-leucyl-L- Formulation,arginyl-L-tryptophyl-L-prolyl-L-tryptophyl- dermal, topicalL-tryptophyl-L-prolyl-L-tyrptophyl-L- Peptidearginyl-L-arginyl, pentahydrochloride antibiotic Antiacne 427leuprorelin 6-D-leucine-9-(N-ethyl-L-prolinamide)- Transmucosal,10-deglycinamide- nasal, Menstruation disorders, Anticancer, hormonal,Fertility enhancer 428 delmitide D-Tyrosinamide, D-arginyl-D-norleucyl-GI inflamma- acetate D-norleucyl-D-norleucyl-D-arginyl-D- tory/bowelnorleucyl-D-norleucyl-D- disorders, norleucylglycyl-, monoacetateRadio/chemo- protective, Antipsoriasis, Antipruritic/in-flamm, allergic, Multiple sclerosis treatment, Alimentary/Meta-bolic, other, Antiviral, anti- HIV, Antiasthma, COPD treatment,Respiratory Stomatological 196 cat PADMRGALLVLALLVTQALGVKMAETCPIFYDVFFAVANGNELLLDL AntiasthmaSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSK Antiallergic,DCMGEAVQNTVEDLKLNTLGR non-asthma 429 NOV-002bis-(gamma-L-glutamyl)-L-cysteinyl-bis- Anticancer, glycin disodium saltimmunological Radio/chemo- sensitizer Antidote 430 GPG-NH2glycyl-prolyl-glycine amide Antiviral, anti- HIV 431 ABT-510NAc-Sar-Gly-ValDalloleThrNValleArgProNHE Anticancer, other   8 CJC-1131GLP-1 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Antidiabetic 432desmopressin Vasopressin, 1-(3-mercaptopropanoic acid)- Formulation,8-D-arginine- oral, Hormone, Antidiabetic, Urological 197 metastinMNSLVSWQLLLFLCATHFGEPLEDVASVGNSRPTGQQLESLGLL Anticancer,APGEQSLPCTERKPAATARLSRRGTSLSPPPESSGSPQQPGLSA otherPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRFGKREAAPGNHG RSAGRG 433 leuprorelin5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L- Anticancerseryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-N-ethyl-L-prolinamide acetate (salt) 434 SGS-111N-phenylacetylprolylglycine ethyl ester Cognition enhancerNeuroprotective 435 taltobulin (4S)-4-[[(2S)-3,3-dimethyl-2-[[(2S)-3-Anticancer, methyl-2-(methylamino)-3- otherphenylbutanoyl]amino]butanoyl]methylamino]- 2,5-dimethylhex-2-enoic acid436 leuprolide 6-D-leucine-9-(N-ethyl-L-prolinamide)- inhalable,10-deglycinamide- systemic, Anticancer, Menstruation disorders 103XOMA-629 gi|157276599|ref|NP_001716.2| Antiacnebactericidal/permeability-increasing Anti-infective,protein precursor [Homo sapiens] otherMRENMARGPCNAPRWASLMVLVAIGTAVTAAVNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITCSSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCEKNTNSVSSELQPYFQTLPVMTKIDSVAGINYGLVAPPATTAETLDVQMKGEFYSENHHNPPPFAPPVMEFPAAHDRMVYLGLSDYFFNTAGLVYQEAGVLKMTLRDDMIPKESKFRLTTKFFGTFLPEVAKKFPNMKIQIHVSASTPPHLSVQPTGLTFYPAVDVQAFAVLPNSSLASLFLIGMHTTGSMEVSAESNRLVGELKLDRLLLELKHSNIGPFPVELLQDIMNYIVPILVLPRVNEKLQKGFPLPTPARVQLYNVVLQPHQNFLLFGADV VYK 198 syntheticgi|8393713|ref|NP_058651.1| Sep (O- Antianaemic erythropoiesisphosphoserine) tRNA: Sec (selenocysteine) Radio/chemo- protRNA synthase isoform 1 [Homo sapiens] protectiveMSTSYGCFWRRFIHGIGRSGDISAVQPKAAGSSLLNKITNSLVLDIIKLAGVHTVANCFVVPMATGMSLTLCFLTLRHKRPKAKYIIWPRIDQKSCFKSMITAGFEPVVIENVLEGDELRTDLKAVEAKVQELGPDCILCIHSTTSCFAPRVPDRLEELAVICANYDIPHIVNNAYGVQSSKCMHLIQQGARVGRIDAFVQSLDKNFMVPVGGAIIAGFNDSFIQEISKMYPGRASASPSLDVLITLLSLGSNGYKKLLKERKEMFSYLSNQIKKLSEAYNERLLHTPHNPISLAMTLKTLDEHRDKAVTQLGSMLFTKQVSGARVVPLGSMQTVSGYTFRGFMSHTNNYPCAYLNAASAIGMKMQDVDLFINRLDRCLKAVRKERSKESDDNYDK TEDVDIEEMALKLDNVLLDTYQDASS191 β-amyloid beta- gi|8176533|gb|AAB26264.2| beta-amyloid Cognitionvaccine amyloid peptide precursor; beta APP [Homo sapiens] enhancerpeptide GSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIIITLVMLKKQYTSNHHGVVE 437 sincalide1-De-(5-oxo-L-proline)-2-de-L- Imaging agentglutamine-5-L-methioninecaerulein Alimentary/Meta- bolic 438 albiglutide([8-glycine]human glucagon-like peptide 1- Antidiabetic(7-36)-peptidyl)([8-glycine]human glucagon- Anorectic/Antio-like peptide 1-(7-36)-peptidyl)(human serum besityalbumin (585 residues)) 199 SB-144 gi|13899257|ref|NP_113622.1|transmembrane Anticancer, and ubiquitin-like domain containing 1 other[Homo sapiens] Radio/chemo- MTLIEGVGDEVTVLFSVLACLLVLALAWVSTHTAEGGDPLPQPSsensitizer GTPTPSQPSAAMAATDSMRGEAPGAETPSLRHRGQAAQPEPSTGFTATPPAPDSPQEPLVLRLKFLNDSEQVARAWPHDTIGSLKRTQFPGREQQVRLIYQGQLLGDDTQTLGSLHLPPNCVLHCHVSTRVGPPNPPCPPGSEPGPSGLEIGSLLLPLLLLLLLLLWYCQIQYRPF FPLTATLGLAGFTLLLSLLAFAMYRP200 exenatide LAR L-histidylglycyl-L-glutamylglycyl-L- Antidiabeticthreonyl-L-phenylalanyl-L-threonyl-L-seryl-L-aspartyl-L-leucyl-L-seryl-L-lysyl-L-glutaminyl-L-methionyl-L-glutamyl-L-glutamyl-L-glutamyl-L-alanyl-L-valyl-L-arginyl-L-leucyl-L-phenylalanyl-L-isoleucyl-L-glutamyl-L-tryptophyl-L-leucyl-L-lysyl-L-asparaginylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-L-prolyl- L-prolyl-L-prolyl-L-serinamide201 BA-058 PTHrP gi|131542|sp|P12272.1|PTHR_HUMAN OsteoporosisParathyroid hormone-related protein treatmentprecursor (PTH-rP) (PTHrP) [Contains:PTHrP[1-36]; PTHrP[38-94]; Osteostatin (PTHrP[107-139])]MQRRLVQQWSVAVFLLSYAVPSCGRSVEGLSRRLKRAVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSRR H   8 BIM-51077 GLP-1HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Antidiabetic[(aminoisobutyric acid) 8,35]hGLP-1(1-36)NH₂, has the same amino acid sequence ashuman GLP-1(7-36 amide) except for thereplacement of amino acids 8 and 35 withα-aminoisobutyric acid (Aib) to reduce protease susceptibility. 202TM-701 H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His- Anticancer,Gln-Met-Ala-Arg-Lys-Cys-Asp-Asp-Cys-Cys-Gly- otherGly-Lys-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln- Radio/chemo-Cys-Leu-Cys-Arg-NH₂ sensitizer(Disulfide bridge: 2-19, 5-28, 16-33, 20-35) 439 CZEN-002[dNal(2′)-7,Phe-12]-α-MSH 6-13 Antifungal, Antibacterial, other,Antiviral, anti- HIV, Immuno- suppressant, Metabolic and enzymedisorders, Anti- inflammatory, Antiarthritic, other GI inflamma-tory/bowel disorders 203 ZP-120 Ac-RYYRWKKKKKKK-NH₂ Cardiostimulant 204CTT H-Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys-OH Formulation technology205 PYY3-36 gi|71361686|ref|NP_004151.2| peptide YY Anorectic/Antio-[Homo sapiens] besity MVFVRRPWPALTTVLLALLVCLGALVDAYPIKPEAPREDASPEELNRYYASLRHYLNLVTRQRYGKRDGPDTLLSKTFFPDGEDRPVR SRSEGPDLW AEZS-130EP1572 UMV1843 [Aib-DTrp-DgTrp-CHO] Growth hormone AnabolicMusculoskeletal 206 AL-108 H-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-OHNeuroprotective Cognition enhancer Antiparkinsonian Ophthalmological 202TM-801 H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His- Imaging agentGln-Met-Ala-Arg-Lys-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Lys-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln- Cys-Leu-Cys-Arg-NH₂(Disulfide bridge: 2-19, 5-28, 16-33, 20-35) 202 TM-901H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His- Anticancer,Gln-Met-Ala-Arg-Lys-Cys-Asp-Asp-Cys-Cys-Gly- otherGly-Lys-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln- Imaging agentCys-Leu-Cys-Arg-NH₂ (Disulfide bridge: 2-19, 5-28, 16-33, 20-35) 440S-0373 TRH pyroGlu-His-Pro-NH₂ (or 5-oxo-L-prolyl- NeurologicalL-histidyl-L-prolinamide) Psychostimulant Antiparkinsonian 205 PYY3-36gi|71361686|ref|NP_004151.2| peptide YY Formulation, [Homo sapiens]oral, other MVFVRRPWPALTTVLLALLVCLGALVDAYPIKPEAPREDASPEEAnorectic/Antio- LNRYYASLRHYLNLVTRQRYGKRDGPDTLLSKTFFPDGEDRPVR besitySRSEGPDLW 207 XG-101 gi|4885433|ref|NP_005447.1| mitogen- Immunologicalactivated protein kinase 8 interacting Cardiovascularprotein 1 [Homo sapiens] NeuroprotectiveMAERESGGLGGGAASPPAASPFLGLHIASPPNFRLTHDISLEEF Immuno-EDEDLSEITDECGISLQCKDTLSLRPPRAGLLSAGGGGAGSRLQ suppressantAEMLQMDLIDATGDTPGAEDDEEDDDEERAARRPGAGPPKAESGQEPASRGQGQSQGQSQGPGSGDTYRPKRPTTLNLFPQVPRSQDTLNNNSLGKKHSWQDRVSRSSSPLKTGEQTPPHEHICLSDELPPQSGPAPTTDRGTSTDSPCRRSTATQMAPPGGPPAAPPGGRGHSHRDRIHYQADVRLEATEEIYLTPVQRPPDAAEPTSAFLPPTESRMSVSSDPDPAAYPSTAGRPHPSISEEEEGFDCLSSPERAEPPGGGWRGSLGEPPPPPRASLSSDTSALSYDSVKYTLVVDEHAQLELVSLRPCFGDYSDESDSATVYDNCASVSSPYESAIGEEYEEAPRPQPPACLSEDSTPDEPDVHFSKKFLNVFMSGRSRSSSAESFGLFSCIINGEEQEQTHRAIFRFVPRHEDELELEVDDPLLVELQAEDYWYEAYNMRTGARGVFPAYYAIEVTKEPEHMAALAKNSDWVDQFRVKFLGSVQVPYHKGNDVLCAAMQKIATTRRLTVHFNPPSSCVLEISVRGVKIGVKADDSQEAKGNKCSHFFQLKNISFCGYHPKNNKYFGFITKHPADHRFACHVFVSEDSTKALAESVGRAFQQFYKQFVEYTCP TEDIYLE 208 XG-102gi|4885433|ref|NP_005447.1| mitogen- Neuroprotectiveactivated protein kinase 8 interacting Cardiovascularprotein 1 [Homo sapiens] OtologicalMAERESGGLGGGAASPPAASPFLGLHIASPPNFRLTHDISLEEF OphthalmologicalEDEDLSEITDECGISLQCKDTLSLRPPRAGLLSAGGGGAGSRLQ AntiparkinsonianAEMLQMDLIDATGDTPGAEDDEEDDDEERAARRPGAGPPKAESG Immuno-QEPASRGQGQSQGQSQGPGSGDTYRPKRPTTLNLFPQVPRSQDT suppressantLNNNSLGKKHSWQDRVSRSSSPLKTGEQTPPHEHICLSDELPPQSGPAPTTDRGTSTDSPCRRSTATQMAPPGGPPAAPPGGRGHSHRDRIHYQADVRLEATEEIYLTPVQRPDAAEPTSAFLPPTESRMSVSSDPDPAAYPSTAGRPHPSISEEEEGFDCLSSPERAEPPGGGWRGSLGEPPPPPRASLSSDTSALSYDSVKYTLVVDEHAQLELVSLRPCFGDYSDESDSATVYDNCASVSSPYESAIGEEYEEAPRPQPPACLSEDSTPDEPDVHFSKKFLNVFMSGRSRSSSAESFGLFSCIINGEEQEQTHRAIFRFVPRHEDELELEVDDPLLVELQAEDYWYEAYNMRTGARGVFPAYYAIEVTKEPEHMAALAKNSDWVDQFRVKFLGSVQVPYHKGNDVLCAAMQKIATTRRLTVHFNPPSSCVLEISVRGVKGVKADDSQEAKGNKCSHFFQLKNISFCGYHPKNNKYFGFITKHPADHRFACHVFVSEDSTKALAESVGRAFQQFYKQFVEYTCPTE DIYLE 441 lanreotide SRL-Threonamide,3-(2-naphthalenyl)-D-alanyl-L- Formulation,cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl- modified-L-valyl-L-cysteinyl-, cyclic (2-7)- release, other disulfideSomatostatin Anti- hypertensive, other 209 OGP-(10-14)-LH-Tyrosine-Glycine-Phenylalanine- Haematological Glycine-Glycine-OHMusculoskeletal 210 WP9QY cyclo(Tyr-Cys-Trp-Ser-Gln-Tyr-Leu-Cys-Antiarthritic, Tyr); cyclo(tyrosyl-cysteinyl- othertryptophyl-seryl-glutaminyl-tyrosyl- Anti- leucyl-cysteinyl-tyrosyl)inflammatory 211 aviptadil His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Anti- Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr- hypertensive,Leu-Asn-Ser-Ile-Leu-Asn other Respiratory Immuno- suppressant 212 AL-209Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala Neuroprotective Cognition enhancerOphthalmological 442 octreotideL-Cysteinamide, D-phenylalanyl-L-cysteinyl- Formulation,L-phenylalanyl-D-tryptophyl-L-lysyl-L- implant threonyl-N-[2-hydroxy-1-Formulation, (hydroxymethyl)propyl]-, cyclic (2-7)- modified-disulfide, [R-(R*,R*)]- release, >24 hr Somatostatin 213 CDX-110Leu-Glu-Glu-Lys-Lys-Gly-Asn-Tyr-Val-Val-Thr- Recombinant Asp-His-Cys-KLHvaccine Anticancer, immunological 444 desmopressinVasopressin, 1-(3-mercaptopropanoic Hormone, Uro- acid)-8-D-arginine-logical, Reproductive/go- nadal, general 445 obinepitide[34-L-glutamine]pancreatic hormone Anorectic/Antio- (human) besityInsulin Insulin (ox), 8A-L-threonine-10A-L- solubility-isoleucine-30B-L-threonine- enhanced Insulin 171 terlipressinN-(N-(N-glycylglycyl)gylcyl)-8-L- Hepato-lysinevasopressin [CAS}; Gly-Gly-Gly-8-Lys- protective,vasopressin; N-(alpha)-glycyl-glycyl- Urological, Giglycyl-8-lysine vasopressin; Remestyp; bleedingTGLVP; glipressin; glycylpressin;glypressin; terlypressin; triglycyl lysine vasopressin; triglycyl-(8-lysine)vasopressin; triglycylvasopressin; vasopressin, tri-Gly-8-Lys-214 ZT-153 Asn-Phe-Gly-Ala-Ile-Leu; NFGAIL; asparagyl- Antidiabeticphenylalanyl-glycyl-alanyl-isoleucyl-leucine; islet amyloid polypeptide (22-27) 215, 215 FGLLgi|42544189|ref|NP_004458.3| Cognition and 216fibrinogen-like 1 precursor [Homo sapiens] enhancerMAKVFSFILVTTALTMGREISALEDCAQEQMRLRAQVRLLETRV NeurologicalKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFI PNVIgi|42544200|ref|NP_963846.1| fibrinogen-like 1 precursor [Homo sapiens]MAKVFSFILVTTALTMGREISALEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFI PNVIgi|42544198|ref|NP_671736.2| fibrinogen-like 1 precursor [Homo sapiens]MAKVFSFILVTTALTMGREISALEDCAQEQMRLRAQVRLLETRV KQQQVIKIQLLQENEVQFLD 217ST-03 gi|386634|gb|AAB27460.1| Recombinant 01-ST-3 =heat-stable enterotoxin growth factor[Vibrio cholerae, 01, Peptide, 19 aa] MusculoskeletalNLIDCCEICCNPACFGCLN Osteoporosis treatment 446 cetrorelixD-Alaninamide, N-acetyl-3-(2- Formulation, acetatenaphthalenyl)-D-alanyl-4-chloro-D- modified-phenylalanyl-3-(3-pyridinyl)-D-alanyl- release, >24 hrL-seryl-L-tyrosyl-N5-(aminocarbonyl)-D- Menstruationol-L-leucyl-L-arginyl-L-prolyl- disorders 218 neuro-alpha toxin, Naja; cobra alpha toxin; Cognition degenerativecobra toxin alpha; toxin alpha, cobra; enhancer thergi|64054|emb|CAA26373.1| unnamed proteinproduct [Laticauda semifasciata]MKTLLLTLVVVTIVCLDLGYTRICFNHQSSQPQTTKTCSPGESSCYNKQWSDFRGTIIERGCGCPTVKPGIKLSCCESEVCNN gi|4519816|dbj|BAA75752.1|short chain neurotoxin [Laticauda semifasciata]MKTLLLTLVVVTIVCLDLGYTRICFNHQSSQPQTTKTCSPGESSCYNKQWSDFRGTIIERGCGCPTVKPGIKLSCCESEVCNN gi|32140561|dbj|BAC78199.1|erabutoxin a [Laticauda semifasciata]MKTLLLTLVVVTIVCLDLGYTRICFNHQSSQPQTTKTCSPGESSCYNKQWSDFRGTIIERGCGCPTVKPGIKLSCCESEVCNN gi|32140563|dbj|BAC78200.1|erabutoxin a [Laticauda semifasciata]MKTLLLTLVVVTIVCLDLGYTRICFNHQSSQPQTTKTCSPGESSCYNKQWSDFRGTIIERGCGCPTVKPGIKLSCCESEVCNN 219 CT-319MSNKKIIKIIKLQIPGGKANPAPPIGPALGAAGVNIMGFCKEFN Antiviral, anti-AATQDRPGDLLPVVITVYSDKTFSFVMKQSPVSSLIKKALGLES HIVGSKIPNRNKVGKLTRAQITVIAEQKMKDMDVVLLESAERMVEGT ARSMGVDVE 447 Peptide TL-Threonine, N-(N-(N2-(N-(N-(N-(N-D-alanyl- AntipsoriasisL-seryl)-L-threonyl)-L-threonyl)-L- Multiplethreonyl)-L-asparaginyl)-L-tyrosyl)- sclerosis[CAS]; HIV Peptide T; Peptide T, HIV treatment Cognition enhancerMusculoskeletal 220 and APP-018 pallidin [Mus musculus] Hypolipaemic/221 gi|9790039|ref|NP_062762.1|[9790039] Anti-MSVPEPPPPDGVLTGPSDSLEAGEPTPGLSDTSPDEGLIEDFPV atherosclerosisDDRAVEHLVGGLLSHYLPDLQRSKRALQELTQNQVVLLDTLEQEISKFKECHSMLDINALFTEAKHYHAKLVTIRKEMLLLHEKTSKLKKRALKLQQKRQREELEREQQREKEFEREKQLTAKPAKRT envelope glycoprotein [Humanimmunodeficiency virus type 1] gi|4205319|gb|AAD11044.1|[4205319]KLTPLCVTLNCTDLDLRNTTNNTTTEERGEMKNCSFNITTNIRDRYQKEYALFYKLDVIPIKEDNTSDNTSYRLISCNTSVITQACPK IS 222 somatropingi|60651145|gb|AAX31661.1| somatotropin Formulation, [Bubalus bubalis]transmucosal, AFPAMSLSSLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYS nasalIQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLG Growth hormonePLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTP AnabolicRAGQILKRTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYL Reproductive/go-RVMKCRRFGEASCAF nadal, general 448 heparin6-[5-acetamido-4,6-dihydroxy-2- Formulation,(sulfooxymethyl)oxan-3-yl]oxy-3-[5-(6- transmucosal,carboxy-4,5-dihydroxy-3-sulfooxyoxan-2- nasalyl)oxy-6-(hydroxymethyl)-3- Anticoagulant(sulfoamino)-4-sulfooxyoxan-2-yl]oxy-4-hydroxy-5-sulfooxyoxane-2-carboxylic acid  46 CGRP CGRPACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH₂ Cardiovascular Cardiostimulant449 YM-216391 A concise total synthesis of the unusual Anticancer,oxazole-based cyclopeptide structure otherYM-216391, which also establishes thestereochemistry of the natural product i.e.1, is described. The unusual polyoxazole-thiazole-based cyclopeptide 1, designatedYM-216391, was recently isolated fromStreptomyces nobilis.1 It shares both astructural and biological homology with thepotent telomerase inhibitor telomestatin 2which is showing promise in cancerchemotherapy.2 The structure of YM-216391comprises a continuum of five azoles whichhave their origins in serine, cysteine andphenylalanine, linked via a glycine-valine-isoleucine tripeptide tether. The completestereochemical assignment of YM-216391 hasnot been established. In this communicationwe describe a concise total synthesis ofthe cyclopeptide, which not only confirmsits unique structure but also allows theassignment of its stereochemistry, shown informula 1. Thus, the 2,4-disubstitutedoxazoles 3 and 4 and the trisubstituted oxazole 5 were first elaborated223 FGLm LSENDEWTQDRAKP Cognition enhancer Neurological 224 prohaninNPFPTWRKRPG Analgesic, other 225 heart failure NPgi|189079|gb|AAA36355.1| natriuretic Cardiostimulant therapy peptideMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNGLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLR RH 450 SEN-304D-[(chG)Y-(chG)(chG)(MeL)]-NH₂, where CognitionchG is R-cyclohexylglycine enhancer Anti- inflammatory 451 PrimacollSynthetic growth factor Musculoskeletal 452 OctreotideL-Cysteinamide, D-phenylalanyl-L-cysteinyl- Formulation,L-phenylalanyl-D-tryptophyl-L-lysyl-L- modified-threonyl-N-[2-hydroxy-1- release, >24 hr(hydroxymethyl)propyl]-, cyclic (2-7)- Symptomaticdisulfide, [R-(R*,R*)]- antidiabetic Ophthalmological Somatostatin 453ALS-02 Glycine, N-(aminoiminomethyl)-N-methyl- Neuroprotective 200exendin-4, GLP-1 L-histidylglycyl-L-glutamylglycyl-L- AntidiabeticPC-DAC threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-aspartyl-L-leucyl-L-seryl-L-lysyl-L-glutaminyl-L-methionyl-L-glutamyl-L-glutamyl-L-glutamyl-L-alanyl-L-valyl-L-arginyl-L-leucyl-L-phenylalanyl-L-isoleucyl-L-glutamyl-L-tryptophyl-L-leucyl-L-lysyl-L-asparaginylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-L-prolyl- L-prolyl-L-prolyl-L-serinamide226 Exenatide gi|1916067|gb|AAB51130.1| exendin 4 Formulation,[Heloderma suspectum] transmucosal,MKIILWLCVFGLFLATLFPISWQMPVESGLSSEDSASSESFASK nasalIKRHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG Antidiabetic 225 Cardeva BNPgi|113836|sp|P16860.1|ANFB_HUMAN Natriuretic Cardiostimulantpeptides B precursor [Contains: Gamma-brainnatriuretic peptide; Brain natriuretic peptide 32 (BNP-32)]MDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLR RH 227 AlloferonH-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly- Immunomodulator,Val-His-Gly-OH anti-infective 454 PAC-G31PAMCF-I; Alveolar Macrophage Chemotactic RecombinantFactor I; Alveolar Macrophage Chemotactic interleukinFactor-I; Anionic Neutrophil Activating RespiratoryPeptide; Anionic Neutrophil-Activating AntiasthmaPeptide; CXCL8 Chemokine; CXCL8 Chemokines; COPD treatmentCXCL8, Chemokine; Chemokine CXCL8; Chemokine, CXCL8; Chemokines, CXCL8;Chemotactic Factor, Macrophage Derived;Chemotactic Factor, Macrophege-Derived;Chemotactic Factor, Neutrophil; ChemotacticFactor, Neutrophil, Monocyte-Derived; Chemotactic Peptide-Interleukin-8,Granulocyte; Granulocyte Chemotactic Peptide Interleukin 8; GranulocyteChemotactic Peptide-Interleukin-8; IL-8;IL8; Interleukin 8; Lymphocyte-DerivedNeutrophil-Activating Peptide; Macrophage-Derived Chemotactic Factor; Monocyte-Derived Neutrophil Chemotactic Factor;Monocyte-Derived Neutrophil-ActivatingPeptide; Neutrophil Activating Peptide,Lymphocyte Derived; Neutrophil ActivatingPeptide, Monocyte Derived; NeutrophilActivation Factor; Neutrophil ChemotacticFactor; Neutrophil-Activating Peptide,Anionic; Neutrophil-Activating Peptide 228 PAC-525 Ac-KWRRWVRWI-NH₂Antibacterial, other 229, 229 PAC-113Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly- Antifungal and 230 Tyrhistatin 10, human; histatin 11, human;histatin 12, human; histatin 3, human;histatin 4, human; histatin 5, human;histatin 6, human; histatin 7, human;histatin 8, human; histatin 9, human;histatin-3 (1-24), human; histatin-3 (1-25),human; histatin-3 (12-24), human; histatin-3(12-25), human; histatin-3 (12-32), human;histatin-3 (13-25), human; histatin-3(5-11), human; histatin-3 (5-12), human;lysyl-phenylalanyl-histidyl-glutamyl-lysyl-histidyl-histidyl-seryl-histidyl-arginyl- glycyl-tyrosinegi|4557653|ref|NP_000191.1| histatin 3 [Homo sapiens]MKFFVFALILALMLSMTGADSHAKRHHGYKRKFHEKHHSHRGYR SNYLYDN 231 MLIFMet-Gln-Cys-Asn-Ser Anti- U.S. Pat. No. 6,524,591 inflammatory 454carfilzomib L-Phenylalaninamide, (alphaS)-alpha-[(4- Anticancer,morpholinylacetyl)amino]benzenebutanoyl- otherL-leucyl-N-[(1S)-3-methyl-1-[[(2R)-2- methyloxiranyl]carbonyl]butyl]-232 NAFB001 gi|63025222|ref|NP_000651.3| transforming Ophthalmologicalgrowth factor, beta 1 [Homo sapiens] HepatoprotectiveMPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYSLNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS [PIR] 233 IL12-NGRH-Cys-Asn-Gly-Arg-Cys-Gly-OH Recombinant, (Disulfide bridge: 1-5) otherCytokine Anticancer, immunological 234 and enterostatinVal-Pro-Val-Asp; Val-Pro-Asp-Pro-Arg Anorectic/Antio- 235 besity 455octreotide L-Cysteinamide, D-phenylalanyl-L-cysteinyl- Formulation,L-phenylalanyl-D-tryptophyl-L-lysyl-L- modified-threonyl-N-[2-hydroxy-1- release, >24 hr(hydroxymethyl)propyl]-, cyclic (2-7)- Somatostatindisulfide, [R-(R*,R*)]- 150 enfuvirtideL-Phenylalaninamide, N-acetyl-L-tyrosyl-L- Formulation,threonyl-L-seryl-L-leucyl-L-isoleucyl-L- parenteral,histidyl-L-seryl-L-leucyl-L-isoleucyl-L- needle-freealpha-glutamyl-L-alpha-glutamyl-L-seryl-L- Antiviral, anti-glutaminyl-L-asparaginyl-L-glutaminyl-L- HIVglutaminyl-L-alpha-glutamyl-L-lysyl-L-asparaginyl-L-alpha-glutamyl-L-glutaminyl-L-alpha-glutamyl-L-leucyl-L-leucyl-L-alpha-glutamyl-L-leucyl-L-alpha-aspartyl-L-lysyl-L-tryptophyl-L-alanyl-L-seryl-L-leucyl-L-tryptophyl-L-asparaginyl-L-trpytophyl- 236 PR-21gi|2213924|gb|AAB61615.1| neural cell Neurologicaladhesion molecule [Homo sapiens] CognitionMLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVGESKFFLCQVA enhancerGDAKDKDISWFSPNGEKLTPNQQRISVVWNDDSSSTLTIYNANIDDAGIYKCVVTGEDGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAVIVCDVVSSLPPTIIWKHKGRDVILKKDVRFIFLSNNYLPIPGIKKTDEGTYRCEGRILARGEINFNDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFPGPTMSWTKDGEQIEQEEHDEKYLFSDDSSHLTIKKVDKNHEAENICIAENKVGEQDATIHLKVFAKPQITYVEDQTAMELAEQVILTVEASGDHIPYITWWTSTWQI 237 AC-163794 GIPgi|183221|gb|AAA53192.1| gastric Antidiabeticinhibitory polypeptide precursorMVATKTFALLLLSLFLAVGLGEKKEGHFSALPSLPVGSHAKVSSPQPRGPRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKNHITQREARALELASQANRKEEEAVEPQSSPAKNPSDEDLLRDL LIQELLACLLDQTNLCRLRSR; 456glucagon Glucagon (1-29); Glukagon; HG Factor; Formulation,HG-Factor; Hyperglycemic Glycogenolytic transdermal,Factor; Hyperglycemic-Glycogenolytic Factor; systemicProglucagon (33-61) hypoglycemia 457 InsulinInsulin (ox), 8A-L-threonine-10A-L- Formulation,isoleucine-30B-L-threonine- oral, other Formulation, optimized,nanoparticles Antidiabetic 458 Dekafin-2DNA Synthesis Factor; Fibroblast Growth Anticancer,Factor; Fibroblast Growth Regulatory otherFactor; Growth Factor, Fibroblast; Growth Factors, Fibroblast 238 andrelaxin (1) Glu-Leu-Tyr-Ser-Ala-Leu-Ala.Asn-Lys-Cys- Recombinant 239Cys-His-Val-Gly-Cys-Thr-Lys-Arg-Ser-Leu-Ala- hormone Arg-Phe-Cys Hormone(2) H-Asp-Ser-Trp-Met-Glu-Glu-Val-Ile-Lys- Labour inducerLeu-Cys-Gly-Arg-Glu-Leu-Val-Arg-Ala-Gln-Ile- Anti-Ala-Ile-Cys-Gly-Met-Ser-Thr-Ser hypertensive,Cys 11 of each chain form disulfide bond; othercys 24 of the first chain forms disulfide bond with cys 23 of chain 2459 rhNRG-1 Differentiation Factor, neu; GGF Protein; Recombinant,Glial Growth Factor; Heregulin; NDF Protein; otherNRG1 Protein; Neuregulin 1; neu Cardiostimulant Differentiation Factor240 c-peptide C-peptide Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Symptomatic analogue Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-antidiabetic Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln 241 SB-101gi|30353933|gb|AAH52287.1| CD44 protein Recombinant, [Homo sapiens]other MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSI Anticancer,SRTEAADLCKAFNSTLPTMAQMEKALSIGFETCSST other 242 Britistatingi|66270695|gb|AAY43681.1| disintegrin Antithromboticisoform D-1 [Bitis arietans]SPPVCGNKILEQGEDCDCGSPANCQDRCCNAATCKLTPGSQCNYGECCDQCRFKKAGTVCRIARGDWNDDYCTGKSSDCPWNH 243 echistatingi|208338|gb|AAA72777.1| echistatin AntithromboticMECESGPCCRNCKFLKEGTICKRARGDDLDDYCNGKTCDCPRNP HKGPAT 244 gastringi|4503923:20-101 gastrin preproprotein diabetes [Homo sapiens]EASWKPRSQQPDAPLGTGANRDLELPWLEQQGPASHHRRQLGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDFGRRSAEDEN 245 herpes simplexgi|9629447:1-23 envelope glycoprotein D Prophylactic vaccine[Human herpesvirus 1] vaccine MGGAAARLGAVILFVVIVGLHGV 246 neurotensingi|5453816:152-163 neurotensin/neuromedin N Analgesic, otherpreproprotein [Homo sapiens] LYENKPRRPYIL 247 nociceptingi|5453922|ref|NP_006219.1| Neurological prepronociceptin [Homo sapiens]Cognition MKVLLCDLLLLSLFSSVFSSCQRDCLTCQEKLHPALDSFDLEVC enhancerILECEEKVFPSPLWTPCTKVMARSSWQLSPAAPEHVAAALYQPR Analgesic, otherASEMQHLRRMPRVRSLFQEQEEPEPGMEEAGEMEQKQLQKRFGGFTGARKSARKLANQKRFSEFMRQYLVLSMQSSQRRRTLHQNGNV 248 oxyntomodulinsp|P01275.3|GLUC_HUMAN:53-89 Glucagon Obesity;precursor [Contains: Glicentin; AntiulcerGlicentin-related polypeptide (GRPP); Oxyntomodulin (OXY) (OXM)]HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA 249 pancreastatingi|164417:256-304 chromogranin A Antidiabetic precursorGWPQAPAMDGAGKTGAEEAQPPEGKGAREHSRQEEEEETAGAPQ GLFRG 250 relaxin Relaxingi|5902052|ref|NP_008842.1| relaxin 1 Recombinantpreproprotein [Homo sapiens] harmoneMPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELVRAQI HormoneAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETIIIML Labour inducerEFIANLPPELKAALSERQPSLPELQQYVPALKDSNLSFEEFKKLIRNRQSEAADSNPSELKYLGLDTHSQKKRRPYVALFEKCCLIGC TKRSLAKYC 251 secretingi|11345450:28-54 secretin Haemostatic; preproprotein [Homo sapiens]diagnostic of HSDGTFTSELSRLREGARLQRLLQGLV pancreatic dysfunction,asthma, COPD, others 252 TIMPMAPFEPLASGILLLLWLIAPSRACTCVPPHPQTAFCNSDLVIRA Recombinant,KFVGTPEVNQTTLYQRYEIKMTKMYKGFQALGDAADIRFVYTPA otherMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWNSLS VulneraryLAQRRGFTKTYTVGCEECTVFPCLSIPCKLQSGTHCLWTDQLLQ Antiarthritic,GSEKGFQSRHLACLPREPGLCTWQSLRSQIA other Stomatological 252 TIMPMAPFEPLASGILLLLWLIAPSRACTCVPPHPQTAFCNSDLVIRA Recombinant,KFVGTPEVNQTTLYQRYEIKMTKMYKGFQALGDAADIRFVYTPA otherMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWNSLS Antiarthritic,LAQRRGFTKTYTVGCEECTVFPCLSIPCKLQSGTHCLWTDQLLQ otherGSEKGFQSRHLACLPREPGLCTWQSLRSQIA Stomatological 253 tendamistatAsp-Thr-Thr-Val-Ser-Glu-Pro-Ala-Pro-Ser-Cys- AntidiabeticVal-Thr-Leu-Tyr-Gln-Ser-Trp-Arg-Tyr-Ser-Gln-Ala-Asp-Asn-Gly-Cys-Ala-Gln-Thr-Val-Thr-Val-Lys-Val-Val-Tyr-Glu-Asp-Asp-Thr-Glu-Gly-Leu-Cys-Tyr-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr-Thr-Val-Gly-Asp-Gly-Tyr-Ile-Gly-Ser-His-Gly-His-Ala-Arg-Tyr-Leu-Ala-Arg-Cys-Leu 254 thymosic β4gi|11056061|ref|NP_066932.1| thymosin, Vulnerary beta 4 [Homo sapiens]Ophthalmological MSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESSymptomatic antidiabetic Dermatological Cardiovascular Septic shocktreatment Antiasthma 255 urodilatinThr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys- CardiostimulantPhe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln- UrologicalSer-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr Antiasthma 256 PharmaprojectsGly-Ser-Arg-Ala-His-Ser-Ser-His-Leu-Lys Anticancer, No. 6236 otherAntiarrhythmic Anti- parkinsonian Cognition enhancer Neuroprotective 257ANUP-1 Glu-Leu-Lys-Cys-Tyr-Thr-Cys-Lys-Glu-Pro-Met- Anticancer,Thr-Ser-Ala-Ala-Cys other 258 DMI-4983 Asp-Ala-His-Lys Cardiovascular460 Glypromate Gly-Pro-Glu Neuroprotective 259 CD-NPLys Met Val Gln Gly Ser Gly Cys Phe Gly Arg CardiostimulantLys Met Asp Ile Ser Ser Ser Ser Gly Leu GlyCys Pro Ser Leu Arg Asp Pro Arg Pro Asn Ala Pro Ser Thr Ser Ala 260Kisspeptin-54 GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPN CancerYNWNSFGLRF-NH2 metastasis, angiogenesis 261 Kisspeptin-14DLPNYNWNSFGLRF-NH2 Cancer metastasis, angiogenesis 262 Kisspeptin-13LPNYNWNSFGLRF-NH2 Cancer metastasis, angiogenesis 263 Kisspeptin-10YNWNSFGLRF-NH2 Cancer metastasis, angiogenesis 264 ZiconotideCKGKGAKCSRLMYDCCTGSCRSGKC 461 BiphalinTyr-D-Ala-Gly-Phe-NH—NH-Phe-Gly-D-Ala-Tyr  39 Nesiritide BrainSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH Netriur- itic peptide (BNP)  40 CD-NPGLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA 265 Protegrin-1 CytolyticRGGRLCYCRRRFCVCVGR-NH2 antibiotic 266 V681Ac-KWKSFLKTFKSAVKTVLHTALKAISS-NH2 462 V681 (V13A_(D))Ac-KWKSFLKTFKSA(AD)KTVLHTALKAISS-NH2 ‘(AD)’ discloses the D-configuration of Alanine 267 V681 des A12 KWKSFLKTFKSVKTVLHTALKAISS 268V681 V13K KWKSFLKTFKSAKKTVLHTALKAISS 269 V681 V13K, T15KKWKSFLKTFKSAKKKVLHTALKAISS 270 GLP-2 GLPHADGSFSDEMNTILDNLAARDFINWLIQTKITD 271 GLP-2 (A2G) GLPHGDGSFSDEMNTILDNLAARDFINWLIQTKITD 272 GLP-2 (A2G/C34) GLPHGDGSFSDEMNTILDNLAARDFINWLIQTKITDC 273 AOD-9604 Human LRIVQCASVEGSCGFYMusculoskeletal, Growth COPD, Hypnotic/ Hormone Sedative,Immunostimulant, Antidiabetic, Anabolic, Symptomatic antidiabetic,Vulnerary 274 Ac-AOD- Human Ac-LRIVQCAKVEGSCGFY Musculoskeletal,9604(S8K) Growth COPD, Hypnotic/ Hormone Sedative, Immunostimulant,Antidiabetic, Anabolic, Symptomatic antidiabetic, Vulnerary 275 Ac-AOD-Human Ac-LRIVQCASVEGSCGFYK Musculoskeletal, 9604(K17) GrowthCOPD, Hypnotic/ Hormone Sedative, Immunostimulant, Antidiabetic,Anabolic, Symptomatic antidiabetic, Vulnerary 276 C-peptide InsulinEAEDLQVGQVELGGGPGAGSLQPLALEGSLQ 463 CR845 Opioidsperipherally-selective kappa opioid receptor agonists

D-Phe-D-Phe-D-Leu-D-Lys-[ω(4- aminopiperidine-4-carboxylic acid)]-OHacute and chronic pain of visceral, inflammatory and neuropathicorigin, and for the treatment of pruritis (itch) 277 Protegrin-2Cytolytic RGGRLCYCRRRFCICV antibiotic 278 Protegrin-3 CytolyticRGGGLCYCRRRFCVCVGRG antibiotic 279 Protegrin-4 CytolyticRGGRLCYCRGWICFCVGRG antibiotic 280 Protegrin-5 CytolyticRGGRLCYCRPRFCVCVGRG antibiotic 281 Preprotegrin CytolyticAVLRAVDRLNEQSSEANLYRLLELDQPPKADEDPGTPKPVSFTVKETVCPRPTRQPPELCDFKENGRVKQCVGTVTLDQIKDPLDITC NEVQGVRGGRLCYCRPRFCVCVGRG248 Oxyntomodulin HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA 276 C-peptideEAEDLQVGQVELGGGPGAGSLQPLALEGSLQ 282 C-peptide EGSLC mutant 283Human Opioid Enkepha- Tyr-Gly-Gly-Phe-Met Growth Factor lin 284cholecystokinin RDY(SO3-)TGW(Nle)DF 285 Dynorphin A YGGFLRRIRPKLK (1-13)464 Pralmorelin D-Ala-D-2-Nal-Ala-Trp-D-Phe-Lys-NH₂ (GHRFA) 286Aniritide RSSCFGGRMDRIGAQSGLGCNSFRY 287 Vessel dilatorEVVPPQVLSDPNEEAGAALSPLPEVPPWTGEVSPAQR proANP31-67 465 Peptide GArg-Pro-Lys-Pro-Gln-Arg-D-Trp-MePhe-D-Trp- Leu-Met 466 TiplimotideD-Ala-lys-pro-val-val-his-leu-phe-ala-asp- ile-val-thr-pro-arg-thr-pro288 Desirudin (63- VVYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGdesulfohirudin) TPKPQSHNDGDFEEIPEEYLQ 467 ExamorelinHis-DTrp(2-Me)-Ala-Trp-DPhe-Lys-NH2 172 Terlipressin Vesopres-Gly-Gly-Gly-c[Cys-Tyr-Phe-Gln-Asn-Cys]-Pro- sin Lys-Gly-NH2 289Osteogenic ALKRQGRTLYGFGG Growth Factor (WT) 290 Osteogenic YGFGGGrowth Factor (10-14) 291 Myelin Basic Ac-ASQKRPSQRHG Protein peptide292 Myelin Basic Ac-ASQYRPSQRHG Protein peptide Ac1-11[4Y] 293Gonadorelin pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (24-33) CONH2468 Bremelanotide Alpha-MSH Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH294 Islet GLHDPSHGTLPNGSG Diabetes Neogenesis associated peptide (INGAP)295 Urocortin II IVLSLDVPIGLLQILLEQARARAAREQATTNARILARVGHC 296 A6 (anti-CH3CO-NH2-KPSSPPEE-CONH2 antiogenic peptide) 297 ObestatinH-Phe-Asn-Ala-Pro-Phe-Asp-Val-Gly-Ile-Lys-Leu-Ser-Gly-Val-Gln-Tyr-Gln-Gln-His-Ser-Gln- Ala-Leu-NH2 298 ITF-1697Gly-Lys(Et)-Pro-Arg 299 CNP (C-type GLSKGCFGLKLDRIGSMSGLGC netriureticpeptide 300 Osteocalcin YLYQWLGAPVPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRDiabetes FYGPV 301 EAEDLQVGQVELGGGPGAGCLQPLALEGSLQ 469 D4F-APO1Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ mimetic peptide

In other embodiments, the therapeutic peptides are selected from thegroup consisting of peptide G, OTS-102, Angiocol (antiangiogenic peptidegroup), ABT-510 (antiangiogenic peptide group), A6 (antiangiogenicpeptide group), islet neogenesis gene associated protein (INGAP),tendamistat, recombinant human carperitide (alpha-atrial natriureticpeptide) (natriuretic peptide group), urodilatin (natriuretic peptidegroup), desirudin, Obestatin, ITF-1697, oxyntomodulin, cholecystokinin,bactericidal permeability increasing (BPI) protein, C-peptide,Prosaptide TX14(A), sermorelin acetate (GHRFA group), pralmorelin (GHRFAgroup), growth hormone releasing factor (GHRFA group), examorelin (GHRFAgroup), gonadorelin (LH-related peptide group), corticoliberin, atrialnatriuretic peptide (natriuretic peptide group), anergix, somatostatin(GHRFA group), 29-amino-acid peptide growth hormone releasing hormone(GHRH) analogue (GHRFA group), bremelanotide (melanocortin agonistgroup), melanocortin peptidomimetic compound (melanocortin agonistgroup), antiprogestogens—GnRH antagonists (LH-related peptide group),recombinant LH (luteinizing hormone) (LH-related peptide group),terlipressin, Ecallantide-60-amino-acid recombinant peptide kallikreininhibitor, calphobindin I, tiplimotide, osteogenic growth peptide,myelin basic protein, dynorphin A, anaritide (natriuretic peptidegroup), secretin, GLP-2, and gastrin.

The therapeutic peptides of the invention may comprise any of the 20natural amino acids, and/or non-natural amino acids, amino acid analogs,and peptidomimetics, in any combination. The peptides may be composed ofD-amino acids or L-amino acids, or a combination of both in anyproportion. In addition to natural amino acids, the therapeutic peptidesmay contain, or may be modified to include, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, or more non-natural amino acids. Exemplary non-naturalamino acids and amino acid analogs that can be use with the inventioninclude, but are not limited to, 2-aminobutyric acid, 2-aminoisobutyricacid, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, 3-methylhistidine,3-pyridylalanine, 4-chlorophenylalanine, 4-fluorophenylalanine,4-hydroxyproline, 5-hydroxylysine, alloisoleucine, citrulline,dehydroalanine, homoarginine, homocysteine, homoserine, hydroxyproline,N-acetylserine, N-formylmethionine, N-methylglycine, N-methylisoleucine,norleucine, N-α-methylarginine, O-phosphoserine, ornithine,phenylglycine, pipecolinic acid, piperazic acid, pyroglutamine,sarcosine, valanine, β-alanine, and β-cyclohexylalanine.

The therapeutic peptides may be, or may be modified to be, linear,branched, or cyclic, with our without branching.

Additionally, the therapeutic peptides may optionally be modified orprotected with a variety of functional groups or protecting groups,including amino terminus protecting groups and/or carboxy terminusprotecting groups. Protecting groups, and the manner in which they areintroduced and removed are described, for example, in “Protective Groupsin Organic Chemistry,” Plenum Press, London, N.Y. 1973; and Greene etal., “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS” 3^(rd) Edition, John Wileyand Sons, Inc., New York, 1999. Numerous protecting groups are known inthe art. An illustrative, non-limiting list of protecting groupsincludes methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl,trifluoroacetyl, N-hydroxysuccinimide, t-butoxycarbonyl, benzoyl,4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl,benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl,4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl,2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl,2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl,and 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl. For discussions ofvarious different types of amino- and carboxy-protecting groups, see,for example, U.S. Pat. No. 5,221,736 (issued Jun. 22, 1993); U.S. Pat.No. 5,256,549 (issued Oct. 26, 1993); U.S. Pat. No. 5,049,656 (issuedSep. 17, 1991); and U.S. Pat. No. 5,521,184 (issued May 28, 1996).

The therapeutic peptides contain, or may be modified to contain,functional groups to which a water-soluble polymer may be attached,either directly or through a spacer moiety or linker. Functional groupsinclude, but are not limited to, the N-terminus of the therapeuticpeptide, the C-terminus of the therapeutic peptide, and any functionalgroups on the side chain of an amino acid, e.g. lysine, cysteine,histidine, aspartic acid, glutamic acid, tyrosine, arginine, serine,methionine, and threonine, present in the therapeutic peptide.

The therapeutic peptides can be prepared by any means known in the art,including non-recombinant and recombinant methods, or they may, in someinstances, be commercially available. Chemical or non-recombinantmethods include, but are not limited to, solid phase peptide synthesis(SPPS), solution phase peptide synthesis, native chemical ligation,intein-mediated protein ligation, and chemical ligation, or acombination thereof. In a preferred embodiment, the therapeutic peptidesare synthesized using standard SPPS, either manually or by usingcommercially available automated SPPS synthesizers.

SPPS has been known in the art since the early 1960's (Merrifield, R.B., J. Am. Chem. Soc., 85:2149-2154 (1963)), and is widely employed.(See also, Bodanszky, Principles of Peptide Synthesis, Springer-Verlag,Heidelberg (1984)). There are several known variations on the generalapproach. (See, for example, “Peptide Synthesis, Structures, andApplications” © 1995 by Academic Press, Chapter 3 and White (2003) FmocSolid Phase Peptide Synthesis, A practical Approach, Oxford UniversityPress, Oxford). Very briefly, in solid phase peptide synthesis, thedesired C-terminal amino acid residue is coupled to a solid support. Thesubsequent amino acid to be added to the peptide chain is protected onits amino terminus with Boc, Fmoc, or other suitable protecting group,and its carboxy terminus is activated with a standard coupling reagent.The free amino terminus of the support-bound amino acid is allowed toreact with the carboxy-terminus of the subsequent amino acid, couplingthe two amino acids. The amino terminus of the growing peptide chain isdeprotected, and the process is repeated until the desired polypeptideis completed. Side chain protecting groups may be utilized as needed.

Alternatively, the therapeutic peptides may be prepared recombinantly.Exemplary recombinant methods used to prepare therapeutic peptidesinclude the following, among others, as will be apparent to one skilledin the art. Typically, a therapeutic peptide as defined and/or describedherein is prepared by constructing the nucleic acid encoding the desiredpeptide or fragment, cloning the nucleic acid into an expression vector,transforming a host cell (e.g., plant, bacteria such as Escherichiacoli, yeast such as Saccharomyces cerevisiae, or mammalian cell such asChinese hamster ovary cell or baby hamster kidney cell), and expressingthe nucleic acid to produce the desired peptide or fragment. Theexpression can occur via exogenous expression or via endogenousexpression (when the host cell naturally contains the desired geneticcoding). Methods for producing and expressing recombinant polypeptidesin vitro and in prokaryotic and eukaryotic host cells are known to thoseof ordinary skill in the art. See, for example, U.S. Pat. No. 4,868,122,and Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold SpringHarbor Laboratory Press (1989).

To facilitate identification and purification of the recombinantpeptide, nucleic acid sequences that encode an epitope tag or otheraffinity binding sequence can be inserted or added in-frame with thecoding sequence, thereby producing a fusion peptide comprised of thedesired therapeutic peptide and a peptide suited for binding. Fusionpeptides can be identified and purified by first running a mixturecontaining the fusion peptide through an affinity column bearing bindingmoieties (e.g., antibodies) directed against the epitope tag or otherbinding sequence in the fusion peptide, thereby binding the fusionpeptide within the column. Thereafter, the fusion peptide can berecovered by washing the column with the appropriate solution (e.g.,acid) to release the bound fusion peptide. Optionally, the tag maysubsequently be removed by techniques known in the art. The recombinantpeptide can also be identified and purified by lysing the host cells,separating the peptide, e.g., by size exclusion chromatography, andcollecting the peptide. These and other methods for identifying andpurifying recombinant peptides are known to those of ordinary skill inthe art.

Related Peptides

It will be appreciated and understood by one of skill in the art thatcertain modifications can be made to the therapeutic peptides definedand/or disclosed herein that do not alter, or only partially abrogate,the properties and activities of these therapeutic peptides. In someinstances, modifications may be made that result in an increase intherapeutic activities. Additionally, modifications may be made thatincrease certain biological and chemical properties of the therapeuticpeptides in a beneficial way, e.g. increased in vivo half life,increased stability, decreased susceptibility to proteolytic cleavage,etc. Thus, in the spirit and scope of the invention, the term“therapeutic peptide” is used herein in a manner to include not only thetherapeutic peptides defined and/or disclosed herein, but also relatedpeptides, i.e. peptides that contain one or more modifications relativeto the therapeutic peptides defined and/or disclosed herein, wherein themodification(s) do not alter, only partially abrogate, or increase thetherapeutic activities as compared to the parent peptide.

Related peptides include, but are not limited to, fragments oftherapeutic peptides, therapeutic peptide variants, and therapeuticpeptide derivatives. Related peptides also include any and allcombinations of these modifications. In a non-limiting example, arelated peptide may be a fragment of a therapeutic peptide as disclosedherein having one or more amino acid substitutions. Thus it will beunderstood that any reference to a particular type of related peptide isnot limited to a therapeutic peptide having only that particularmodification, but rather encompasses a therapeutic peptide having thatparticular modification and optionally any other modification.

Related peptides may be prepared by action on a parent peptide or aparent protein (e.g. proteolytic digestion to generate fragments) orthrough de novo preparation (e.g. solid phase synthesis of a peptidehaving a conservative amino acid substitution relative to the parentpeptide). Related peptides may arise by natural processes (e.g.processing and other post-translational modifications) or may be made bychemical modification techniques. Such modifications are well-known tothose of skill in the art.

A related peptide may have a single alteration or multiple alterationsrelative to the parent peptide. Where multiple alterations are present,the alterations may be of the same type or a given related peptide maycontain different types of modifications. Furthermore, modifications canoccur anywhere in a polypeptide, including the peptide backbone, theamino acid side-chains, and the N- or C-termini.

As previously noted, related peptides include fragments of thetherapeutic peptides defined and/or disclosed herein, wherein thefragment retains some of or all of at least one therapeutic activity ofthe parent peptide. The fragment may also exhibit an increase in atleast one therapeutic activity of the parent peptide. In certainembodiments of the invention, therapeutic peptides include relatedpeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 contiguous amino acidresidues, or more than 125 contiguous amino acid residues, of any of thetherapeutic peptides disclosed, herein, including in Table 1. In otherembodiments of the invention, therapeutic peptides include relatedpeptides having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, or 50 amino acid residues deleted from the N-terminus and/orhaving 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or50 amino acid residues deleted from the C-terminus of any of thetherapeutic peptides disclosed herein, including in Table 1.

Related peptides also include variants of the therapeutic peptidesdefined and/or disclosed herein, wherein the variant retains some of orall of at least one therapeutic activity of the parent peptide. Thevariant may also exhibit an increase in at least one therapeuticactivity of the parent peptide. In certain embodiments of the invention,therapeutic peptides include variants having 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, or 50 conservative and/ornon-conservative amino acid substitutions relative to the therapeuticpeptides disclosed herein, including in Table 1. Desired amino acidsubstitutions, whether conservative or non-conservative, can bedetermined by those skilled in the art.

In certain embodiments of the invention, therapeutic peptides includevariants having conservative amino substitutions; these substitutionswill produce a therapeutic peptide having functional and chemicalcharacteristics similar to those of the parent peptide. In otherembodiments, therapeutic peptides include variants havingnon-conservative amino substitutions; these substitutions will produce atherapeutic peptide having functional and chemical characteristics thatmay differ substantially from those of the parent peptide. In certainembodiments of the invention, therapeutic peptide variants have bothconservative and non-conservative amino acid substitutions. In otherembodiments, each amino acid residue may be substituted with alanine.

Natural amino acids may be divided into classes based on common sidechain properties: nonpolar (Gly, Ala, Val, Leu, Ile, Met); polar neutral(Cys, Ser, Thr, Pro, Asn, Gln); acidic (Asp, Glu); basic (His, Lys,Arg); and aromatic (Tip, Tyr, Phe). By way of example, non-conservativeamino acid substitutions may involve the substitution of an amino acidof one class for that of another, and may be introduced in regions ofthe peptide not critical for therapeutic activity.

Preferably, amino acid substitutions are conservative. Conservativeamino acid substitutions may involve the substitution of an amino acidof one class for that of the same class. Conservative amino acidsubstitutions may also encompass non-natural amino acid residues,including peptidomimetics and other atypical forms of amino acidmoieties, and may be incorporated through chemical peptide synthesis,

Amino acid substitutions may be made with consideration to thehydropathic index of amino acids. The importance of the hydropathicamino acid index in conferring interactive biological function on aprotein is generally understood in the art (Kyte et al., 1982, J. Mol.Biol. 157:105-31). Each amino acid has been assigned a hydropathic indexon the basis of its hydrophobicity and charge characteristics. Thehydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsbiological properties. According to U.S. Pat. No. 4,554,101,incorporated herein by reference, the following hydrophilicity valueshave been assigned to these amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

In certain embodiments of the invention, therapeutic peptides includevariants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, or 50 amino acid deletions relative to the therapeutic peptidesdisclosed herein, including in Table 1. The deleted amino acid(s) may beat the N- or C-terminus of the peptide, at both termini, at an internallocation or locations within the peptide, or both internally and at oneor both termini. Where the variant has more than one amino aciddeletion, the deletions may be of contiguous amino acids or of aminoacids at different locations within the primary amino acid sequence ofthe parent peptide.

In other embodiments of the invention, therapeutic peptides includevariants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, or 50 amino acid additions relative to the therapeutic peptidesdisclosed herein, including in Table 1. The added amino acid(s) may beat the N- or C-terminus of the peptide, at both termini, at an internallocation or locations within the peptide, or both internally and at oneor both termini. Where the variant has more than one amino acidaddition, the amino acids may be added contiguously, or the amino acidsmay be added at different locations within the primary amino acidsequence of the parent peptide.

Addition variants also include fusion peptides. Fusions can be madeeither at the N-terminus or at the C-terminus of the therapeuticpeptides disclosed herein, including in Table 1. In certain embodiments,the fusion peptides have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, or 50 amino acid additions relative to the therapeuticpeptides disclosed herein, including in Table 1. Fusions may be attacheddirectly to the therapeutic peptide with no connector molecule or may bethrough a connector molecule. As used in this context, a connectormolecule may be an atom or a collection of atoms optionally used to linka therapeutic peptide to another peptide. Alternatively, the connectormay be an amino acid sequence designed for cleavage by a protease toallow for the separation of the fused peptides.

The therapeutic peptides of the invention may be fused to peptidesdesigned to improve certain qualities of the therapeutic peptide, suchas therapeutic activity, circulation time, or reduced aggregation.Therapeutic peptides may be fused to an immunologically active domain,e.g. an antibody epitope, to facilitate purification of the peptide, orto increase the in vivo half life of the peptide. Additionally,therapeutic peptides may be fused to known functional domains, cellularlocalization sequences, or peptide permeant motifs known to improvemembrane transfer properties.

In certain embodiments of the invention, therapeutic peptides alsoinclude variants incorporating one or more non-natural amino acids,amino acid analogs, and peptidomimetics. Thus the present inventionencompasses compounds structurally similar to the therapeutic peptidesdefined and/or disclosed herein, which are formulated to mimic the keyportions of the therapeutic peptides of the present invention. Suchcompounds may be used in the same manner as the therapeutic peptides ofthe invention. Certain mimetics that mimic elements of protein secondaryand tertiary structure have been previously described. Johnson et al.,Biotechnology and Pharmacy, Pezzuto et al. (Eds.), Chapman and Hall, NY,1993. The underlying rationale behind the use of peptide mimetics isthat the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions.A peptide mimetic is thus designed to permit molecular interactionssimilar to the parent peptide. Mimetics can be constructed to achieve asimilar spatial orientation of the essential elements of the amino acidside chains. Methods for generating specific structures have beendisclosed in the art. For example, U.S. Pat. Nos. 5,446,128, 5,710,245,5,840,833, 5,859,184, 5,440,013; 5,618,914, 5,670,155, 5,475,085,5,929,237, 5,672,681 and 5,674,976, the contents of which are herebyincorporated by reference, all disclose peptidomimetics structures thatmay have improved properties over the parent peptide, for example theymay be conformationally restricted, be more thermally stable, exhibitincreased resistance to degredation, etc.

In another embodiment, related peptides comprise or consist of a peptidesequence that is at least 70% identical to any of the therapeuticpeptides disclosed herein, including in Table 1. In additionalembodiments, related peptides are at least 75% identical, at least 80%identical, at least 85% identical, 90% identical, at least 91%identical, at least 92% identical, 93% identical, at least 94%identical, at least 95% identical, 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to any ofthe therapeutic peptides disclosed herein, including in Table 1.

Sequence identity (also known as % homology) of related polypeptides canbe readily calculated by known methods. Such methods include, but arenot limited to those described in Computational Molecular Biology (A. M.Lesk, ed., Oxford University Press 1988); Biocomputing: Informatics andGenome Projects (D. W. Smith, ed., Academic Press 1993); ComputerAnalysis of Sequence Data (Part 1, A. M. Griffin and H. G. Griffin,eds., Humana Press 1994); G. von Heinle, Sequence Analysis in MolecularBiology (Academic Press 1987); Sequence Analysis Primer (M. Gribskov andJ. Devereux, eds., M. Stockton Press 1991); and Carillo et al., 1988,SIAM J. Applied Math., 48:1073.

Preferred methods to determine sequence identity and/or similarity aredesigned to give the largest match between the sequences tested. Methodsto determine sequence identity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,1990, J. Mol. Biol. 215:403-10). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda,Md.); Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span,” asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 0.1× the gap opening penalty), as well as a comparison matrixsuch as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.A standard comparison matrix is also used by the algorithm (see Dayhoffet al., 5 Atlas of Protein Sequence and Structure (Supp. 3 1978)(PAM250comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA89:10915-19 (BLOSUM 62 comparison matrix)). The particular choices to bemade with regard to algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, and thresholds of similarity will bereadily apparent to those of skill in the art and will depend on thespecific comparison to be made.

Related peptides also include derivatives of the therapeutic peptidesdefined and/or disclosed herein, wherein the variant retains some of orall of at least one therapeutic activity of the parent peptide. Thederivative may also exhibit an increase in at least one therapeuticactivity of the parent peptide. Chemical alterations of therapeuticpeptide derivatives include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. (See, for instance, T. E.Creighton, Proteins, Structure and Molecular Properties, 2nd ed., W.H.Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, ed., Academic Press, New York,pgs. 1-12 (1983); Seifter et al., Meth. Enzymol 182:626-46 (1990);Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62, 1992).

Therapeutic peptide derivatives also include molecules formed by thedeletion of one or more chemical groups from the parent peptide. Methodsfor preparing chemically modified derivatives of the therapeuticpeptides defined and/or disclosed herein are known to one of skill inthe art.

In some embodiments of the invention, the therapeutic peptides may bemodified with one or more methyl or other lower alkyl groups at one ormore positions of the therapeutic peptide sequence. Examples of suchgroups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,pentyl, etc. In certain preferred embodiments, arginine, lysine, andhistidine residues of the therapeutic peptides are modified with methylor other lower alkyl groups.

In other embodiments of the invention, the therapeutic peptides may bemodified with one or more glycoside moieties relative to the parentpeptide. Although any glycoside can be used, in certain preferredembodiments the therapeutic peptide is modified by introduction of amonosaccharide, a disaccharide, or a trisaccharide or it may contain aglycosylation sequence found in natural peptides or proteins in anymammal. The saccharide may be introduced at any position, and more thanone glycoside may be introduced. Glycosylation may occur on a naturallyoccurring amino acid residue in the therapeutic peptide, oralternatively, an amino acid may be substituted with another formodification with the saccharide.

Glycosylated therapeutic peptides may be prepared using conventionalFmoc chemistry and solid phase peptide synthesis techniques, e.g., onresin, where the desired protected glycoamino acids are prepared priorto peptide synthesis and then introduced into the peptide chain at thedesired position during peptide synthesis. Thus, the therapeutic peptidepolymer conjugates may be conjugated in vitro. The glycosylation mayoccur before deprotection. Preparation of aminoacid glycosides isdescribed in U.S. Pat. No. 5,767,254, WO 2005/097158, and Doores, K., etal., Chem. Commun., 1401-1403, 2006, which are incorporated herein byreference in their entireties. For example, alpha and beta selectiveglycosylations of serine and threonine residues are carried out usingthe Koenigs-Knorr reaction and Lemieux's in situ anomerizationmethodology with Schiff base intermediates. Deprotection of the Schiffbase glycoside is then carried out using mildly acidic conditions orhydrogenolysis. A composition, comprising a glycosylated therapeuticpeptide conjugate made by stepwise solid phase peptide synthesisinvolving contacting a growing peptide chain with protected amino acidsin a stepwise manner, wherein at least one of the protected amino acidsis glycosylated, followed by water-soluble polymer conjugation, may havea purity of at least 95%, such as at least 97%, or at least 98%, of asingle species of the glycosylated and conjugated therapeutic peptide.

Monosaccharides that may by used for introduction at one or more aminoacid residues of the therapeutic peptides defined and/or disclosedherein include glucose (dextrose), fructose, galactose, and ribose.Additional monosaccharides suitable for use include glyceraldehydes,dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose,xylose, ribulose, xylulose, allose, altrose, mannose, N-Acetylneuraminicacid, fucose, N-Acetylgalactosamine, and N-Acetylglucosamine, as well asothers. Glycosides, such as mono-, di-, and trisaccharides for use inmodifying a therapeutic peptide, may be naturally occurring or may besynthetic. Disaccharides that may by used for introduction at one ormore amino acid residues of the therapeutic peptides defined and/ordisclosed herein include sucrose, lactose, maltose, trehalose,melibiose, and cellobiose, among others. Trisaccharides includeacarbose, raffinose, and melezitose.

In further embodiments of the invention, the therapeutic peptidesdefined and/or disclosed herein may be chemically coupled to biotin. Thebiotin/therapeutic peptide molecules can then to bind to avidin.

As previously noted, modifications may be made to the therapeuticpeptides defined and/or disclosed herein that do not alter, or onlypartially abrogate, the properties and activities of these therapeuticpeptides. In some instances, modifications may be made that result in anincrease in therapeutic activity. Thus, included in the scope of theinvention are modifications to the therapeutic peptides disclosedherein, including in Table 1, that retain at least 1%, at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,and any range derivable therein, such as, for example, at least 70% toat least 80%, and more preferably at least 81% to at least 90%; or evenmore preferably, between at least 91% and at least 99% of thetherapeutic activity relative to the unmodified therapeutic peptide.Also included in the scope of the invention are modification to thetherapeutic peptides disclosed herein, including in Table 1, that havegreater than 100%, greater than 110%, greater than 125%, greater than150%, greater than 200%, or greater than 300%, or greater than 10-foldor greater than 100-fold, and any range derivable therein, of thetherapeutic activity relative to the unmodified therapeutic peptide.

The level of therapeutic activity of a given therapeutic peptide, or amodified therapeutic peptide, may be determined by any suitable in vivoor in vitro assay. For example, therapeutic activity may be assayed incell culture, or by clinical evaluation, EC₅₀ assays, IC₅₀ assays, ordose response curves. In vitro or cell culture assays, for example, arecommonly available and known to one of skill in the art for manytherapeutic peptides as disclosed herein, including in Table 1. It willbe understood by one of skill in the art that the percent activity of amodified therapeutic peptide relative to its unmodified parent can bereadily ascertained through a comparison of the activity of each asdetermined through the assays disclosed herein or as known to one ofskill in the art.

One of skill in the art will be able to determine appropriatemodifications to the therapeutic peptides defined and/or disclosedherein, including those disclosed herein, including in Table 1. Foridentifying suitable areas of the therapeutic peptides that may bechanged without abrogating their therapeutic activities, one of skill inthe art may target areas not believed to be essential for activity. Forexample, when similar peptides with comparable activities exist from thesame species or across other species, one of skill in the art maycompare those amino acid sequences to identify residues that areconserved among similar peptides. It will be understood that changes inareas of a therapeutic peptide that are not conserved relative tosimilar peptides would be less likely to adversely affect thethereapeutic activity. One skilled in the art would also know that, evenin relatively conserved regions, one may substitute chemically similaramino acids while retaining therapeutic activity. Therefore, even areasthat may be important for biological activity and/or for structure maybe subject to amino acid substitutions without destroying thetherapeutic activity or without adversely affecting the peptidestructure.

Additionally, as appropriate, one of skill in the art can reviewstructure-function studies identifying residues in similar peptides thatare important for activity or structure. In view of such a comparison,one can predict the importance of an amino acid residue in a therapeuticpeptide that corresponds to an amino acid residue that is important foractivity or structure in similar peptides. One of skill in the art mayopt for amino acid substitutions within the same class of amino acidsfor such predicted important amino acid residues of the therapeuticpeptides.

Also, as appropriate, one of skill in the art can also analyze thethree-dimensional structure and amino acid sequence in relation to thatstructure in similar peptides. In view of such information, one of skillin the art may predict the alignment of amino acid residues of atherapeutic peptide with respect to its three dimensional structure. Oneof skill in the art may choose not to make significant changes to aminoacid residues predicted to be on the surface of the peptide, since suchresidues may be involved in important interactions with other molecules.Moreover, one of skill in the art may generate variants containing asingle amino acid substitution at each amino acid residue for testpurposes. The variants could be screened using therapeutic activityassays known to those with skill in the art. Such variants could be usedto gather information about suitable modifications. For example, where achange to a particular amino acid residue resulted in abrogated,undesirably reduced, or unsuitable activity, variants with such amodification would be avoided. In other words, based on informationgathered from routine experimentation, one of skill in the art canreadily determine the amino acids where further modifications should beavoided either alone or in combination with other modifications.

One of skill in the art may also select suitable modifications based onsecondary structure predication. A number of scientific publicationshave been devoted to the prediction of secondary structure. See Moult,1996, Curr. Opin. Biotechnol. 7:422-27; Chou et al., 1974, Biochemistry13:222-45; Chou et al., 1974, Biochemistry 113:211-22; Chou et al.,1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47:45-48; Chou et al., 1978,Ann. Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys. J.26:367-84. Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two peptides orproteins which have a sequence identity of greater than 30%, orsimilarity greater than 40%, often have similar structural topologies.Recent growth of the protein structural database (PDB,http://www.rcsb.org/pdb/home/home.do) has provided enhancedpredictability of secondary, tertiary, and quarternary structure,including the potential number of folds within the structure of apeptide or protein. See Holm et al., 1999, Nucleic Acids Res. 27:244-47.It has been suggested that there are a limited number of folds in agiven peptide or protein and that once a critical number of structureshave been resolved, structural prediction will become dramatically moreaccurate (Brenner et al., 1997, Curr. Opin. Struct. Biol. 7:369-76).

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science,253:164-70; Gribskov et al., 1990, Methods Enzymol. 183:146-59; Gribskovet al., 1987, Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and“evolutionary linkage” (See Holm et al., supra, and Brenner et al.,supra).

Therapeutic Peptide Conjugates

As described above, a conjugate of the invention comprises awater-soluble polymer covalently attached (either directly or through aspacer moiety or linker) to a therapeutic peptide. Typically, for anygiven conjugate, there will be about one to five water-soluble polymerscovalently attached to a therapeutic peptide (wherein for eachwater-soluble polymer, the water-soluble polymer can be attached eitherdirectly to the therapeutic peptide or through a spacer moiety).

To elaborate, a therapeutic peptide conjugate of the invention typicallyhas about 1, 2, 3, or 4 water-soluble polymers individually attached toa therapeutic peptide. That is to say, in certain embodiments, aconjugate of the invention will possess about 4 water-soluble polymersindividually attached to a therapeutic peptide, or about 3 water-solublepolymers individually attached to a therapeutic peptide, or about 2water-soluble polymers individually attached to a therapeutic peptide,or about 1 water-soluble polymer attached to a therapeutic peptide. Thestructure of each of the water-soluble polymers attached to thetherapeutic peptide may be the same or different. One therapeuticpeptide conjugate in accordance with the invention is one having awater-soluble polymer releasably attached to the therapeutic peptide,particularly at the N-terminus of the therapeutic peptide. Anothertherapeutic peptide conjugate in accordance with the invention is onehaving a water-soluble polymer stably attached to the therapeuticpeptide, particularly at the N-terminus of the therapeutic peptide.Another therapeutic peptide conjugate is one having a water-solublepolymer releasably attached to the therapeutic peptide, particularly atthe C-terminus of the therapeutic peptide. Another therapeutic peptideconjugate in accordance with the invention is one having a water-solublepolymer stably attached to the therapeutic peptide, particularly at theC-terminus of the therapeutic peptide. Other therapeutic peptideconjugates in accordance with the invention are those having awater-soluble polymer releasably or stably attached to an amino acidwithin the therapeutic peptide. Additional water-soluble polymers may bereleasably or stably attached to other sites on the therapeutic peptide,e.g., such as one or more additional sites. For example, a therapeuticpeptide conjugate having a water-soluble polymer releasably attached tothe N-terminus may additionally possess a water-soluble polymer stablyattached to a lysine residue. In one embodiment, one or more amino acidsmay be inserted, at the N- or C-terminus, or within the peptide toreleasably or stably attach a water soluble polymer. One preferredembodiment of the present invention is a mono-therapeutic peptidepolymer conjugate, i.e., a therapeutic peptide having one water-solublepolymer covalently attached thereto. In an even more preferredembodiment, the water-soluble polymer is one that is attached to thetherapeutic peptide at its N-terminus.

Preferably, a therapeutic peptide polymer conjugate of the invention isabsent a metal ion, i.e., the therapeutic peptide is not chelated to ametal ion.

For the therapeutic peptide polymer conjugates described herein, thetherapeutic peptide may optionally possess one or more N-methylsubstituents. Alternatively, for the therapeutic peptide polymerconjugates described herein, the therapeutic peptide may beglycosylated, e.g., having a mono- or disaccharide, ornaturally-occurring amino acid glycosylation covalently attached to oneor more sites thereof.

As discussed herein, the compounds of the present invention may be madeby various methods and techniques known and available to those skilledin the art.

The Water-Soluble Polymer

A conjugate of the invention comprises a therapeutic peptide attached,stably or releasably, to a water-soluble polymer. The water-solublepolymer is typically hydrophilic, nonpeptidic, and biocompatible. Asubstance is considered biocompatible if the beneficial effectsassociated with use of the substance alone or with another substance(e.g., an active agent such a therapeutic peptide) in connection withliving tissues (e.g., administration to a patient) outweighs anydeleterious effects as evaluated by a clinician, e.g., a physician. Asubstance is considered nonimmunogenic if the intended use of thesubstance in vivo does not produce an undesired immune response (e.g.,the formation of antibodies) or, if an immune response is produced, thatsuch a response is not deemed clinically significant or important asevaluated by a clinician. Typically, the water-soluble polymer ishydrophilic, biocompatible and nonimmunogenic.

Further the water-soluble polymer is typically characterized as havingfrom 2 to about 300 termini, preferably from 2 to 100 termini, and morepreferably from about 2 to 50 termini. Examples of such polymersinclude, but are not limited to, poly(alkylene glycols) such aspolyethylene glycol (PEG), poly(propylene glycol) (“PPG”), copolymers ofethylene glycol and propylene glycol and the like, poly(oxyethylatedpolyol), poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), andcombinations of any of the foregoing, including copolymers andterpolymers thereof.

The water-soluble polymer is not limited to a particular structure andmay possess a linear architecture (e.g., alkoxy PEG or bifunctionalPEG), or a non-linear architecture, such as branched, forked,multi-armed (e.g., PEGs attached to a polyol core), or dendritic (i.e.having a densely branched structure with numerous end groups). Moreover,the polymer subunits can be organized in any number of differentpatterns and can be selected, e.g., from homopolymer, alternatingcopolymer, random copolymer, block copolymer, alternating tripolymer,random tripolymer, and block tripolymer.

One particularly preferred type of water-soluble polymer is apolyalkylene oxide, and in particular, polyethylene glycol (or PEG).Generally, a PEG used to prepare a therapeutic peptide polymer conjugateof the invention is “activated” or reactive. That is to say, theactivated PEG (and other activated water-soluble polymers collectivelyreferred to herein as “polymeric reagents”) used to form a therapeuticpeptide conjugate comprises an activated functional group suitable forcoupling to a desired site or sites on the therapeutic peptide. Thus, apolymeric reagent for use in preparing a therapeutic peptide conjugateincludes a functional group for reaction with the therapeutic peptide.

Representative polymeric reagents and methods for conjugating suchpolymers to an active moiety are known in the art, and are, e.g.,described in Harris, J. M. and Zalipsky, S., eds, Poly(ethylene glycol),Chemistry and Biological Applications, ACS, Washington, 1997; Veronese,F., and J. M Harris, eds., Peptide and Protein PEGylation, Advanced DrugDelivery Reviews, 54(4); 453-609 (2002); Zalipsky, S., et al., “Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, ed., Plenus Press, New York (1992); Zalipsky(1995) Advanced Drug Reviews 16:157-182, and in Roberts, et al., Adv.Drug Delivery Reviews, 54, 459-476 (2002).

Additional PEG reagents suitable for use in forming a conjugate of theinvention, and methods of conjugation are described in the Pasut. G., etal., Expert Opin. Ther. Patents (2004), 14(5). PEG reagents suitable foruse in the present invention also include those available from NOFCorporation, as described generally on the NOF website(http://nofamerica.net/store/). Products listed therein and theirchemical structures are expressly incorporated herein by reference.Additional PEGs for use in forming a therapeutic peptide conjugate ofthe invention include those available from Polypure (Norway) and fromQuantaBioDesign LTD (Ohio), where the contents of their online catalogs(2006) with respect to available PEG reagents are expressly incorporatedherein by reference. In addition, water soluble polymer reagents usefulfor preparing peptide conjugates of the invention can be preparedsynthetically. Descriptions of the water soluble polymer reagentsynthesis can be found in, for example, U.S. Pat. Nos. 5,252,714,5,650,234, 5,739,208, 5,932,462, 5,629,384, 5,672,662, 5,990,237,6,448,369, 6,362,254, 6,495,659, 6,413,507, 6,376,604, 6,348,558,6,602,498, and 7,026,440.

Typically, the weight-average molecular weight of the water-solublepolymer in the conjugate is from about 100 Daltons to about 150,000Daltons. Exemplary ranges include weight-average molecular weights inthe range of from about 250 Daltons to about 80,000 Daltons, from 500Daltons to about 80,000 Daltons, from about 500 Daltons to about 65,000Daltons, from about 500 Daltons to about 40,000 Daltons, from about 750Daltons to about 40,000 Daltons, from about 1000 Daltons to about 30,000Daltons. In a preferred embodiment, the weight average molecular weightof the water-soluble polymer in the conjugate ranges from about 1000Daltons to about 10,000 Daltons. In certain other preferred embodiments,the range is from about 1000 Daltons to about 5000 Daltons, from about5000 Daltons to about 10,000 Daltons, from about 2500 Daltons to about7500 Daltons, from about 1000 Daltons to about 3000 Daltons, from about3000 Daltons to about 7000 Daltons, or from about 7000 Daltons to about10,000 Daltons. In a further preferred embodiment, the weight averagemolecular weight of the water-soluble polymer in the conjugate rangesfrom about 20,000 Daltons to about 40,000 Daltons. In other preferredembodiments, the range is from about 20,000 Daltons to about 30,000Daltons, from about 30,000 Daltons to about 40,000 Daltons, from about25,000 Daltons to about 35,000 Daltons, from about 20,000 Daltons toabout 26,000 Daltons, from about 26,000 Daltons to about 34,000 Daltons,or from about 34,000 Daltons to about 40,000 Daltons.

For any given water-soluble polymer, a molecular weight in one or moreof these ranges is typical. Generally, a therapeutic peptide conjugatein accordance with the invention, when intended for subcutaneous orintravenous administration, will comprise a PEG or other suitablewater-soluble polymer having a weight average molecular weight of about20,000 Daltons or greater, while a therapeutic peptide conjugateintended for pulmonary administration will generally, although notnecessarily, comprise a PEG polymer having a weight average molecularweight of about 20,000 Daltons or less.

Exemplary weight-average molecular weights for the water-soluble polymerinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons,about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons,about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons,about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000Daltons, about 70,000 Daltons, and about 75,000 Daltons.

Branched versions of the water-soluble polymer (e.g., a branched 40,000Dalton water-soluble polymer comprised of two 20,000 Dalton polymers orthe like) having a total molecular weight of any of the foregoing canalso be used. In one or more particular embodiments, depending upon theother features of the subject therapeutic peptide polymer conjugate, theconjugate is one that does not have one or more attached PEG moietieshaving a weight-average molecular weight of less than about 6,000Daltons.

In instances in which the water-soluble polymer is a PEG, the PEG willtypically comprise a number of (OCH₂CH₂) monomers. As used herein, thenumber of repeat units is typically identified by the subscript “n” in,for example, “(OCH₂CH₂)_(n).” Thus, the value of (n) typically fallswithin one or more of the following ranges: from 2 to about 3400, fromabout 100 to about 2300, from about 100 to about 2270, from about 136 toabout 2050, from about 225 to about 1930, from about 450 to about 1930,from about 1200 to about 1930, from about 568 to about 2727, from about660 to about 2730, from about 795 to about 2730, from about 795 to about2730, from about 909 to about 2730, and from about 1,200 to about 1,900.Preferred ranges of n include from about 10 to about 700, and from about10 to about 1800. For any given polymer in which the molecular weight isknown, it is possible to determine the number of repeating units (i.e.,“n”) by dividing the total weight-average molecular weight of thepolymer by the molecular weight of the repeating monomer.

With regard to the molecular weight of the water-soluble polymer, in oneor more embodiments of the invention, depending upon the other featuresof the particular therapeutic peptide conjugate, the conjugate comprisesa therapeutic peptide covalently attached to a water-soluble polymerhaving a molecular weight greater than about 2,000 Daltons.

A polymer for use in the invention may be end-capped, that is, a polymerhaving at least one terminus capped with a relatively inert group, suchas a lower alkoxy group (i.e., a C₁₋₆ alkoxy group) or a hydroxyl group.One frequently employed end-capped polymer is methoxy-PEG (commonlyreferred to as mPEG), wherein one terminus of the polymer is a methoxy(—OCH₃) group. The -PEG- symbol used in the foregoing generallyrepresents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where (n) generally ranges from aboutzero to about 4,000.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, are also suitable for use in the present invention.For example, the PEG may be described generally according to thestructure:

where poly_(a) and poly_(b) are PEG backbones (either the same ordifferent), such as methoxy poly(ethylene glycol); R″ is a non-reactivemoiety, such as H, methyl or a PEG backbone; and P and Q arenon-reactive linkages. In one embodiment, the branched PEG molecule isone that includes a lysine residue, such as the following reactive PEGsuitable for use in forming a therapeutic peptide conjugate. Althoughthe branched PEG below is shown with a reactive succinimidyl group, thisrepresents only one of a myriad of reactive functional groups suitablefor reacting with a therapeutic peptide.

In some instances, the polymeric reagent (as well as the correspondingconjugate prepared from the polymeric reagent) may lack a lysine residuein which the polymeric portions are connected to amine groups of thelysine via a “—OCH₂CONHCH₂CO—” group. In still other instances, thepolymeric reagent (as well as the corresponding conjugate prepared fromthe polymeric reagent) may lack a branched water-soluble polymer thatincludes a lysine residue (wherein the lysine residue is used to effectbranching).

Additional branched-PEGs for use in forming a therapeutic peptideconjugate of the present invention include those described in co-ownedU.S. Patent Application Publication No. 2005/0009988. Representativebranched polymers described therein include those having the followinggeneralized structure:

where POLY¹ is a water-soluble polymer; POLY² is a water-solublepolymer; (a) is 0, 1, 2 or 3; (b) is 0, 1, 2 or 3; (e) is 0, 1, 2 or 3;(f′) is 0, 1, 2 or 3; (g′) is 0, 1, 2 or 3; (h) is 0, 1, 2 or 3; (j) is0 to 20; each R¹ is independently H or an organic radical selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl and substituted aryl; X¹, when present, is aspacer moiety; X², when present, is a spacer moiety; X⁵, when present,is a spacer moiety; X⁶, when present, is a spacer moiety; X⁷, whenpresent, is a spacer moiety; X⁸, when present, is a spacer moiety; R⁵ isa branching moiety; and Z is a reactive group for coupling to atherapeutic peptide, optionally via an intervening spacer. POLY¹ andPOLY² in the preceding branched polymer structure may be different oridentical, i.e., are of the same polymer type (structure) and molecularweight.

A preferred branched polymer falling into the above classificationsuitable for use in the present invention is:

where (m) is 2 to 4000, and (f) is 0 to 6 and (n) is 0 to 20.

Branched polymers suitable for preparing a conjugate of the inventionalso include those represented more generally by the formulaR(POLY)_(y), where R is a central or core molecule from which extends 2or more POLY arms such as PEG. The variable y represents the number ofPOLY arms, where each of the polymer arms can independently beend-capped or alternatively, possess a reactive functional group at itsterminus. A more explicit structure in accordance with this embodimentof the invention possesses the structure, R(POLY-Z)_(y), where each Z isindependently an end-capping group or a reactive group, e.g., suitablefor reaction with a therapeutic peptide. In yet a further embodimentwhen Z is a reactive group, upon reaction with a therapeutic peptide,the resulting linkage can be hydrolytically stable, or alternatively,may be degradable, i.e., hydrolyzable. Typically, at least one polymerarm possesses a terminal functional group suitable for reaction with,e.g., a therapeutic peptide. Branched PEGs such as those representedgenerally by the formula, R(PEG)_(y) above possess 2 polymer arms toabout 300 polymer arms (i.e., n ranges from 2 to about 300). Preferably,such branched PEGs typically possess from 2 to about 25 polymer arms,such as from 2 to about 20 polymer arms, from 2 to about 15 polymerarms, or from 3 to about 15 polymer arms. Multi-armed polymers includethose having 3, 4, 5, 6, 7 or 8 arms.

Core molecules in branched PEGs as described above include polyols,which are then further functionalized. Such polyols include aliphaticpolyols having from 1 to 10 carbon atoms and from 1 to 10 hydroxylgroups, including ethylene glycol, alkane diols, alkyl glycols,alkylidene alkyl diols, alkyl cycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,ducitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose,glucose, fructose, sorbose, mannose, pyranose, altrose, talose,tagitose, pyranosides, sucrose, lactose, maltose, and the like.Additional aliphatic polyols include derivatives of glyceraldehyde,glucose, ribose, mannose, galactose, and related stereoisomers. Othercore polyols that may be used include crown ether, cyclodextrins,dextrins and other carbohydrates such as starches and amylose. Typicalpolyols include glycerol, pentaerythritol, sorbitol, andtrimethylolpropane.

As will be described in more detail in the linker section below,although any of a number of linkages can be used to covalently attach apolymer to a therapeutic peptide, in certain instances, the linkage isdegradable, designated herein as L_(D), that is to say, contains atleast one bond or moiety that hydrolyzes under physiological conditions,e.g., an ester, hydrolyzable carbamate, carbonate, or other such group.In other instances, the linkage is hydrolytically stable.

Illustrative multi-armed PEGs having 3 arms, 4 arms, and 8 arms areknown and are available commercially and/or can be prepared followingtechniques known to those skilled in the art. Multi-armed activatedpolymers for use in the method of the invention include thosecorresponding to the following structure, where E represents a reactivegroup suitable for reaction with a reactive group on the therapeuticpeptide. In one or more embodiments, E is an —OH (for reaction with atherapeutic peptide carboxy group or equivalent), a carboxylic acid orequivalent (such as an active ester), a carbonic acid (for reaction withtherapeutic peptide —OH groups), or an amino group.

In the structure above, PEG is —(CH₂CH₂O)_(n)CH₂CH₂—, and m is selectedfrom 3, 4, 5, 6, 7, and 8. In certain embodiments, typical linkages areester, carboxyl and hydrolyzable carbamate, such that thepolymer-portion of the conjugate is hydrolyzed in vivo to release thetherapeutic peptide from the intact polymer conjugate. In suchinstances, the linker L is designated as L_(D).

Alternatively, the polymer may possess an overall forked structure asdescribed in U.S. Pat. No. 6,362,254. This type of polymer segment isuseful for reaction with two therapeutic peptide moieties, where the twotherapeutic peptide moieties are positioned a precise or predetermineddistance apart.

In any of the representative structures provided herein, one or moredegradable linkages may additionally be contained in the polymersegment, POLY, to allow generation in vivo of a conjugate having asmaller PEG chain than in the initially administered conjugate.Appropriate physiologically cleavable (i.e., releasable) linkagesinclude but are not limited to ester, carbonate ester, carbamate,sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal. Such linkageswhen contained in a given polymer segment will often be stable uponstorage and upon initial administration.

The PEG polymer used to prepare a therapeutic peptide polymer conjugatemay comprise a pendant PEG molecule having reactive groups, such ascarboxyl or amino, covalently attached along the length of the PEGrather than at the end of the PEG chain(s). The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In certain embodiments, a therapeutic peptide polymer conjugateaccording to one aspect of the invention is one comprising a therapeuticpeptide releasably attached, preferably at its N-terminus, to awater-soluble polymer. Hydrolytically degradable linkages, useful notonly as a degradable linkage within a polymer backbone, but also, in thecase of certain embodiments of the invention, for covalently attaching awater-soluble polymer to a therapeutic peptide, include: carbonate;imine resulting, for example, from reaction of an amine and an aldehyde(see, e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3);phosphate ester, formed, for example, by reacting an alcohol with aphosphate group; hydrazone, e.g., formed by reaction of a hydrazide andan aldehyde; acetal, e.g., formed by reaction of an aldehyde and analcohol; orthoester, formed, for example, by reaction between a formateand an alcohol; and esters, and certain urethane (carbamate) linkages.

Illustrative PEG reagents for use in preparing a releasable therapeuticpeptide conjugate in accordance with the invention are described in U.S.Pat. Nos. 6,348,558, 5,612,460, 5,840,900, 5,880,131, and 6,376,470.

Additional PEG reagents for use in the invention include hydrolyzableand/or releasable PEGs and linkers such as those described in U.S.Patent Application Publication No. 2006-0293499. In the resultingconjugate, the therapeutic peptide and the polymer are each covalentlyattached to different positions of the aromatic scaffold, e.g., Fmoc orFMS, structure, and are releasable under physiological conditions.Generalized structures corresponding to the polymers described thereinare provided below.

For example, one such polymeric reagent comprises the followingstructure:

where POLY¹ is a first water-soluble polymer; POLY² is a secondwater-soluble polymer; X¹ is a first spacer moiety; X² is a secondspacer moiety;

is an aromatic-containing moiety bearing an ionizable hydrogen atom,H_(α); R¹ is H or an organic radical; R² is H or an organic radical; and(FG) is a functional group capable of reacting with an amino group of anactive agent to form a releasable linkage, such as a carbamate linkage(such as N-succinimidyloxy, 1-benzotriazolyloxy, oxycarbonylimidazole,—O—C(O)—Cl, O—C(O)—Br, unsubstituted aromatic carbonate radicals andsubstituted aromatic carbonate radicals). The polymeric reagent caninclude one, two, three, four or more electron altering groups attachedto the aromatic-containing moiety.

Preferred aromatic-containing moieties are bicyclic and tricyclicaromatic hydrocarbons. Fused bicyclic and tricyclic aromatics includepentalene, indene, naphthalene, azulene, heptalene, biphenylene,as-indacene, s-indacene, acenaphthylene, fluorene, phenalene,phenanthrene, anthracene, and fluoranthene.

A preferred polymer reagent possesses the following structure,

where mPEG corresponds to CH₃O—(CH₂CH₂O)_(n)CH₂CH₂—, X¹ and X² are eachindependently a spacer moiety having an atom length of from about 1 toabout 18 atoms, n ranges from 10 to 1800, p is an integer ranging from 1to 8, R¹ is H or lower alkyl, R² is H or lower alkyl, and Ar is anaromatic hydrodrocarbon, preferably a bicyclic or tricyclic aromatichydrocarbon. FG is as defined above. Preferably, FG corresponds to anactivated carbonate ester suitable for reaction with an amino group ontherapeutic peptide. Preferred spacer moieties, X¹ and X², include—NH—C(O)—CH₂—O—, —NH—C(O)—(CH₂)_(q)—O—, —NH—C(O)—(CH₂)_(q)—C(O)—NH—,—NH—C(O)—(CH₂)_(q)—, and —C(O)—NH—, where q is selected from 2, 3, 4,and 5. Preferably, although not necessarily, the nitrogen in thepreceding spacers is proximal to the PEG rather than to the aromaticmoiety.

Another such branched (2-armed) polymeric reagent comprised of twoelectron altering groups comprises the following structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R²,

and (FG) is as defined immediately above, and R^(e1) is a first electronaltering group; and R^(e2) is a second electron altering group. Anelectron altering group is a group that is either electron donating (andtherefore referred to as an “electron donating group”), or electronwithdrawing (and therefore referred to as an “electron withdrawinggroup”). When attached to the aromatic-containing moiety bearing anionizable hydrogen atom, an electron donating group is a group havingthe ability to position electrons away from itself and closer to orwithin the aromatic-containing moiety. When attached to thearomatic-containing moiety bearing an ionizable hydrogen atom, anelectron withdrawing group is a group having the ability to positionelectrons toward itself and away from the aromatic-containing moiety.Hydrogen is used as the standard for comparison in the determination ofwhether a given group positions electrons away or toward itself.Preferred electron altering groups include, but are not limited to,—CF₃, —CH₂CF₃, —CH₂C₆F₅, —CN, —NO₂, —S(O)R, —S(O)Aryl, —S(O₂)R,—S(O₂)Aryl, —S(O₂)OR, —S(O₂)OAryl, —S(O₂)NHR, —S(O₂)NHAryl, —C(O)R,—C(O)Aryl, —C(O)OR, —C(O)NHR, and the like, wherein R is H or an organicradical.

An additional branched polymeric reagent suitable for use in the presentinvention comprises the following structure:

where POLY¹ is a first water-soluble polymer; POLY² is a secondwater-soluble polymer; X¹ is a first spacer moiety; X² is a secondspacer moiety; Ar¹ is a first aromatic moiety; Ar² is a second aromaticmoiety; H_(α) is an ionizable hydrogen atom; R¹ is H or an organicradical; R² is H or an organic radical; and (FG) is a functional groupcapable of reacting with an amino group of therapeutic peptide to form areleasable linkage, such as carbamate linkage.

Another exemplary polymeric reagent comprises the following structure:

wherein each of POLY¹, POLY², X¹, X², Ar¹, Ar², H_(α), R¹, R², and (FG)is as previously defined, and R^(e1) is a first electron altering group.While stereochemistry is not specifically shown in any structureprovided herein, the provided structures contemplate both enantiomers,as well as compositions comprising mixtures of each enantiomer in equalamounts (i.e., a racemic mixture) and unequal amounts.

Yet an additional polymeric reagent for use in preparing a therapeuticpeptide conjugate possesses the following structure:

wherein each of POLY¹, POLY², X¹, X², Ar¹, Ar², H_(α), R¹, R², and (FG)is as previously defined, and R^(e1) is a first electron altering group;and R^(e2) is a second electron altering group.

A preferred polymeric reagent comprises the following structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and, as can be seen from the structure above, thearomatic moiety is a fluorene. The POLY arms substituted on the fluorenecan be in any position in each of their respective phenyl rings, i.e.,POLY¹-X¹— can be positioned at any one of carbons 1, 2, 3, and 4, andPOLY²-X²— can be in any one of positions 5, 6, 7, and 8.

Yet another preferred fluorene-based polymeric reagent comprises thefollowing structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andR^(e2) is a second electron altering group as described above.

Yet another exemplary polymeric reagent for conjugating to a therapeuticpeptide comprises the following fluorene-based structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andR^(e2) is a second electron altering group.

Particular fluorene-based polymeric reagents for forming a releasabletherapeutic peptide polymer conjugate in accordance with the inventioninclude the following:

Still another exemplary polymeric reagent comprises the followingstructure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andR^(e2) is a second electron altering group. Branched reagents suitablefor preparing a releasable therapeutic peptide conjugate includeN-{di(mPEG(20,000)oxymethylcarbonylamino)fluoren-9-ylmethoxycarbonyloxy}succinimide, N-[2,7di(4mPEG(10,000)aminocarbonylbutyrylamino)fluoren-9ylmethoxycarbonyloxy]-succinimide (“G2PEG2Fmoc_(20k)-NHS”), andPEG2-CAC-Fmoc_(4k)-BTC. Of course, PEGs of any molecular weight as setforth herein may be employed in the above structures, and the particularactivating groups described above are not meant to be limiting in anyrespect, and may be substituted by any other suitable activating groupsuitable for reaction with a reactive group present on the therapeuticpeptide.

Those of ordinary skill in the art will recognize that the foregoingdiscussion describing water-soluble polymers for use in forming atherapeutic peptide conjugate is by no means exhaustive and is merelyillustrative, and that all polymeric materials having the qualitiesdescribed above are contemplated. As used herein, the term “polymericreagent” generally refers to an entire molecule, which can comprise awater-soluble polymer segment, as well as additional spacers andfunctional groups.

The Linkage

The particular linkage between the therapeutic peptide and thewater-soluble polymer depends on a number of factors. Such factorsinclude, for example, the particular linkage chemistry employed, theparticular spacer moieties utilized, if any, the particular therapeuticpeptide, the available functional groups within the therapeutic peptide(either for attachment to a polymer or conversion to a suitableattachment site), and the possible presence of additional reactivefunctional groups or absence of functional groups within the therapeuticpeptide due to modifications made to the peptide such as methylationand/or glycosylation, and the like.

In one or more embodiments of the invention, the linkage between thetherapeutic peptide and the water-soluble polymer is a releasablelinkage. That is, the water-soluble polymer is cleaved (either throughhydrolysis, an enzymatic processes, or otherwise), thereby resulting inan unconjugated therapeutic peptide. Preferably, the releasable linkageis a hydrolytically degradable linkage, where upon hydrolysis, thetherapeutic peptide, or a slightly modified version thereof, isreleased. The releasable linkage may result in the water-soluble polymer(and any spacer moiety) detaching from the therapeutic peptide in vivo(and in vitro) without leaving any fragment of the water-soluble polymer(and/or any spacer moiety or linker) attached to the therapeuticpeptide. Exemplary releasable linkages include carbonate, carboxylateester, phosphate ester, thiolester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, carbamates, and orthoesters. Such linkagescan be readily formed by reaction of the therapeutic peptide and/or thepolymeric reagent using coupling methods commonly employed in the art.Hydrolyzable linkages are often readily formed by reaction of a suitablyactivated polymer with a non-modified functional group contained withinthe therapeutic peptide. Preferred positions for covalent attachment ofa water-soluble polymer induce the N-terminal, the C-terminal, as wellas the internal lysines. Preferred releasable linkages include carbamateand ester.

Generally speaking, a preferred therapeutic peptide conjugate of theinvention will possess the following generalized structure:

where POLY is a water-soluble polymer such as any of the illustrativepolymeric reagents provided in Tables 2-4 herein, X is a linker, and insome embodiments a hydrolyzable linkage (L_(D)), and k is an integerselected from 1, 2, and 3, and in some instances 4, 5, 6, 7, 8, 9 and10. In the generalized structure above, where X is L_(D), L_(D) refersto the hydrolyzable linkage per se (e.g., a carbamate or an esterlinkage), while “POLY” is meant to include the polymer repeat units,e.g., CH₃(OCH₂CH₂)_(n),—. In a preferred embodiment of the invention, atleast one of the water-soluble polymer molecules is covalently attachedto the N-terminus of therapeutic peptide. In one embodiment of theinvention, k equals 1 and X is —O—C(O)—NH—, where the —NH— is part ofthe therapeutic peptide residue and represents an amino group thereof.

Although releasable linkages are exemplary, the linkage between thetherapeutic peptide and the water-soluble polymer (or the linker moietythat is attached to the polymer) may be a hydrolytically stable linkage,such as an amide, a urethane (also known as carbamate), amine, thioether(also known as sulfide), or urea (also known as carbamide). One suchembodiment of the invention comprises a therapeutic peptide having awater-soluble polymer such as PEG covalently attached at the N-terminusof therapeutic peptide. In such instances, alkylation of the N-terminalresidue permits retention of the charge on the N-terminal nitrogen.

With regard to linkages, in one or more embodiments of the invention, aconjugate is provided that comprises a therapeutic peptide covalentlyattached at an amino acid residue, either directly or through a linkercomprised of one or more atoms, to a water-soluble polymer.

The conjugates (as opposed to an unconjugated therapeutic peptide) mayor may not possess a measurable degree of therapeutic peptide activity.That is to say, a conjugate in accordance with the invention willtypically possess anywhere from about 0% to about 100% or more of thetherapeutic activity of the unmodified parent therapeutic peptide.Typically, compounds possessing little or no therapeutic activitycontain a releasable linkage connecting the polymer to the therapeuticpeptide, so that regardless of the lack of therapeutic activity in theconjugate, the active parent molecule (or a derivative thereof havingtherapeutic activity) is released by cleavage of the linkage (e.g.,hydrolysis upon aqueous-induced cleavage of the linkage). Such activitymay be determined using a suitable in vivo or in vitro model, dependingupon the known activity of the particular moiety having therapeuticpeptide activity employed.

Optimally, cleavage of a linkage is facilitated through the use ofhydrolytically cleavable and/or enzymatically cleavable linkages such asurethane, amide, certain carbamate, carbonate or ester-containinglinkages. In this way, clearance of the conjugate via cleavage ofindividual water-soluble polymer(s) can be modulated by selecting thepolymer molecular size and the type of functional group for providingthe desired clearance properties. In certain instances, a mixture ofpolymer conjugates is employed where the polymers possess structural orother differences effective to alter the release (e.g., hydrolysis rate)of the therapeutic peptide, such that one can achieve a desiredsustained delivery profile.

One of ordinary skill in the art can determine the proper molecular sizeof the polymer as well as the cleavable functional group, depending uponseveral factors including the mode of administration. For example, oneof ordinary skill in the art, using routine experimentation, candetermine a proper molecular size and cleavable functional group byfirst preparing a variety of polymer-(therapeutic peptide) conjugateswith different weight-average molecular weights, degradable functionalgroups, and chemical structures, and then obtaining the clearanceprofile for each conjugate by administering the conjugate to a patientand taking periodic blood and/or urine samples. Once a series ofclearance profiles has been obtained for each tested conjugate, aconjugate or mixture of conjugates having the desired clearanceprofile(s) can be determined.

For conjugates possessing a hydrolytically stable linkage that couplesthe therapeutic peptide to the water-soluble polymer, the conjugate willtypically possess a measurable degree of therapeutic activity. Forinstance, such conjugates are typically characterized as having atherapeutic activity satisfying one or more of the following percentagesrelative to that of the unconjugated therapeutic peptide: at least 2%,at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 100%, more than 105%,more than 10-fold, or more than 100-fold (when measured in a suitablemodel, such as those presented here and/or known in the art). Often,conjugates having a hydrolytically stable linkage (e.g., an amidelinkage) will possess at least some degree of the therapeutic activityof the unmodified parent therapeutic peptide.

Exemplary conjugates in accordance with the invention will now bedescribed. Amino groups on a therapeutic peptide provide a point ofattachment between the therapeutic peptide and the water-solublepolymer. For example, a therapeutic peptide may comprise one or morelysine residues, each lysine residue containing an s-amino group thatmay be available for conjugation, as well as one amino terminus.

There are a number of examples of suitable water-soluble polymericreagents useful for forming covalent linkages with available amines of atherapeutic peptide. Certain specific examples, along with thecorresponding conjugates, are provided in Table 2 below. In the table,the variable (n) represents the number of repeating monomeric units and“PEP” represents a therapeutic peptide following conjugation to thewater-soluble polymer. While each polymeric portion [e.g., (OCH₂CH₂)_(n)or (CH₂CH₂O)_(n)] presented in Table 2 terminates in a “CH₃” group,other groups (such as H and benzyl) can be substituted therefore.

As will be clearly understood by one skilled in the art, for conjugatessuch as those set forth below resulting from reaction with a therapeuticpeptide amino group, the amino group extending from the therapeuticpeptide designation “˜NH-PEP” represents the residue of the therapeuticpeptide itself in which the ˜NH— is an amino group of the therapeuticpeptide. One preferred site of attachment for the polymeric reagentsshown below is the N-terminus. Further, although the conjugates inTables 2-4 herein illustrate a single water-soluble polymer covalentlyattached to a therapeutic peptide, it will be understood that theconjugate structures on the right are meant to also encompass conjugateshaving more than one of such water-soluble polymer molecules covalentlyattached to therapeutic peptide, e.g., 2, 3, or 4 water-soluble polymermolecules.

TABLE 2 Amine-Specific Polymeric Reagents and the Therapeutic PeptideConjugates Formed Therefrom Polymeric Reagent Corresponding Conjugate

Amine Conjugation and Resulting Conjugates

Conjugation of a polymeric reagent to an amine group of a therapeuticpeptide can be accomplished by a variety of techniques. In one approach,a therapeutic peptide is conjugated to a polymeric reagentfunctionalized with an active ester such as a succinimidyl derivative(e.g., an N-hydroxysuccinimide ester). In this approach, the polymericreagent bearing the reactive ester is reacted with the therapeuticpeptide in aqueous media under appropriate pH conditions, e.g., from pHsranging from about 3 to about 8, about 3 to about 7, or about 4 to about6.5. Most polymer active esters can couple to a target peptide such astherapeutic peptide at physiological pH, e.g., at 7.0. However, lessreactive derivatives may require a different pH. Typically, activatedPEGs can be attached to a peptide such as therapeutic peptide at pHsfrom about 7.0 to about 10.0 for covalent attachment to an internallysine. Typically, lower pHs are used, e.g., 4 to about 5.75, forpreferential covalent attachment to the N-terminus. Thus, differentreaction conditions (e.g., different pHs or different temperatures) canresult in the attachment of a water-soluble polymer such as PEG todifferent locations on the therapeutic peptide (e.g., internal lysinesversus the N-terminus). Coupling reactions can often be carried out atroom temperature, although lower temperatures may be required forparticularly labile therapeutic peptide moieties. Reaction times aretypically on the order of minutes, e.g., 30 minutes, to hours, e.g.,from about 1 to about 36 hours), depending upon the pH and temperatureof the reaction. N-terminal PEGylation, e.g., with a PEG reagent bearingan aldehyde group, is typically conducted under mild conditions, pHsfrom about 5-10, for about 6 to 36 hours. Varying ratios of polymericreagent to therapeutic peptide may be employed, e.g., from an equimolarratio up to a 10-fold molar excess of polymer reagent. Typically, up toa 5-fold molar excess of polymer reagent will suffice.

In certain instances, it may be preferable to protect certain aminoacids from reaction with a particular polymeric reagent if site specificor site selective covalent attachment is desired using commonly employedprotection/deprotection methodologies such as those well known in theart.

In an alternative approach to direct coupling reactions, the PEG reagentmay be incorporated at a desired position of the therapeutic peptideduring peptide synthesis. In this way, site-selective introduction ofone or more PEGs can be achieved. See, e.g., International PatentPublication No. WO 95/00162, which describes the site selectivesynthesis of conjugated peptides.

Exemplary conjugates that can be prepared using, for example, polymericreagents containing a reactive ester for coupling to an amino group oftherapeutic peptide, comprise the following alpha-branched structure:

where POLY is a water-soluble polymer, (a) is either zero or one; X¹,when present, is a spacer moiety comprised of one or more atoms; R¹ ishydrogen an organic radical; and “˜NH-PEP” represents a residue of atherapeutic peptide, where the underlined amino group represents anamino group of the therapeutic peptide.

With respect to the structure corresponding to that referred to in theimmediately preceding paragraph, any of the water-soluble polymersprovided herein can be defined as POLY, any of the spacer moietiesprovided herein can be defined as X¹ (when present), any of the organicradicals provided herein can be defined as R¹ (in instances where R¹ isnot hydrogen), and any of the therapeutic peptides provided herein canbe employed. In one or more embodiments corresponding to the structurereferred to in the immediately preceding paragraph, POLY is apoly(ethylene glycol) such as H₃CO(CH₂CH₂O)_(n)—, wherein (n) is aninteger having a value of from 3 to 4000, more preferably from 10 toabout 1800; (a) is one; X¹ is a C₁₋₆ alkylene, such as one selected frommethylene (i.e., —CH₂—), ethylene (i.e., —CH₂—CH₂—) and propylene (i.e.,—CH₂—CH₂—CH₂—); R¹ is H or lower alkyl such as methyl or ethyl; and PEPcorresponds to any therapeutic peptide disclosed herein, including inTable 1.

Typical of another approach for conjugating a therapeutic peptide to apolymeric reagent is reductive amination. Typically, reductive aminationis employed to conjugate a primary amine of a therapeutic peptide with apolymeric reagent functionalized with a ketone, aldehyde or a hydratedform thereof (e.g., ketone hydrate and aldehyde hydrate). In thisapproach, the primary amine from the therapeutic peptide (e.g., theN-terminus) reacts with the carbonyl group of the aldehyde or ketone (orthe corresponding hydroxy-containing group of a hydrated aldehyde orketone), thereby forming a Schiff base. The Schiff base, in turn, isthen reductively converted to a stable conjugate through use of areducing agent such as sodium borohydride or any other suitable reducingagent. Selective reactions (e.g., at the N-terminus) are possible,particularly with a polymer functionalized with a ketone or analpha-methyl branched aldehyde and/or under specific reaction conditions(e.g., reduced pH).

Exemplary conjugates that can be prepared using, for example, polymericreagents containing an aldehyde (or aldehyde hydrate) or ketone or(ketone hydrate) possess the following structure:

where POLY is a water-soluble polymer; (d) is either zero or one; X²,when present, is a spacer moiety comprised of one or more atoms; (b) isan integer having a value of one through ten; (c) is an integer having avalue of one through ten; R², in each occurrence, is independently H oran organic radical; R³, in each occurrence, is independently H or anorganic radical; and “˜NH-PEP” represents a residue of a therapeuticpeptide, where the underlined amino group represents an amino group ofthe therapeutic peptide.

Yet another illustrative conjugate of the invention possesses thestructure:

where k ranges from 1 to 3, and n ranges from 10 to about 1800.

With respect to the structure corresponding to that referred to inimmediately preceding paragraph, any of the water-soluble polymersprovided herein can be defined as POLY, any of the spacer moietiesprovided herein can be defined as X² (when present), any of the organicradicals provided herein can be independently defined as R² and R³ (ininstances where R² and R³ are independently not hydrogen), and any ofthe PEP moieties provided herein can be defined as a therapeuticpeptide. In one or more embodiments of the structure referred to in theimmediately preceding paragraph, POLY is a poly(ethylene glycol) such asH₃CO(CH₂CH₂O)_(n)—, wherein (n) is an integer having a value of from 3to 4000, more preferably from 10 to about 1800; (d) is one; X¹ is amide[e.g., —C(O)NH—]; (b) is 2 through 6, such as 4; (c) is 2 through 6,such as 4; each of R² and R³ are independently H or lower alkyl, such asmethyl when lower alkyl; and PEP is therapeutic peptide.

Another example of a therapeutic peptide conjugate in accordance withthe invention has the following structure:

wherein each (n) is independently an integer having a value of from 3 to4000, preferably from 10 to 1800; X² is as previously defined; (b) is 2through 6; (c) is 2 through 6; R², in each occurrence, is independentlyH or lower alkyl; and “˜NH-PEP” represents a residue of a therapeuticpeptide, where the underlined amino group represents an amino group ofthe therapeutic peptide.

Additional therapeutic peptide polymer conjugates resulting fromreaction of a water-soluble polymer with an amino group of therapeuticpeptide are provided below. The following conjugate structures arereleasable. One such structure corresponds to:

where mPEG is CH₃O—(CH₂CH₂O)_(n)CH₂CH₂—, n ranges from 10 to 1800, p isan integer ranging from 1 to 8, R¹ is H or lower alkyl, R² is H or loweralkyl, Ar is an aromatic hydrocarbon, such as a fused bicyclic ortricyclic aromatic hydrocarbon, X¹ and X² are each independently aspacer moiety having an atom length of from about 1 to about 18 atoms,˜NH-PEP is as previously described, and k is an integer selected from 1,2, and 3. The value of k indicates the number of water-soluble polymermolecules attached to different sites on the therapeutic peptide. In apreferred embodiment, R¹ and R² are both H. The spacer moieties, X¹ andX², preferably each contain one amide bond. In a preferred embodiment,X¹ and X² are the same. Preferred spacers, i.e., X¹ and X², include—NH—C(O)—CH₂—O—, —NH—C(O)—(CH₂)_(q)—O—, —NH—C(O)—(CH₂)_(q)—C(O)—NH—,—NH—C(O)—(CH₂)_(q)—, and —C(O)—NH—, where q is selected from 2, 3, 4,and 5. Although the spacers can be in either orientation, preferably,the nitrogen is proximal to the PEG rather than to the aromatic moiety.Illustrative aromatic moieties include pentalene, indene, naphthalene,indacene, acenaphthylene, and fluorene.

Particularly preferred conjugates of this type are provided below.

Additional therapeutic peptide conjugates resulting from covalentattachment to amino groups of therapeutic peptide that are alsoreleasable include the following:

where X is either —O— or —NH—C(O)—, Ar₁ is an aromatic group, e.g.,ortho, meta, or para-substituted phenyl, and k is an integer selectedfrom 1, 2, and 3. Particular conjugates of this type include:

where n ranges from about 10 to about 1800.

Additional releasable conjugates in accordance with the invention areprepared using water-soluble polymer reagents such as those described inU.S. Pat. No. 6,214,966. Such water-soluble polymers result in areleasable linkage following conjugation, and possess at least onereleasable ester linkage close to the covalent attachment to the activeagent. The polymers generally possess the following structure,PEG-W—CO₂—NHS or an equivalent activated ester, where

W═—O₂C—(CH₂)_(b)—O— b=1-5

—O—(CH₂)_(b)CO₂—(CH₂)_(c) — b=1-5, c=2-5

—O—(CH₂)_(b)—CO₂—(CH₂)_(c)—O— b=1-5, c=2-5

and NHS is N-hydroxysuccinimidyl. Upon hydrolysis, the resultingreleased active agent, e.g., therapeutic peptide, will possess a shorttag resulting from hydrolysis of the ester functionality of the polymerreagent. Illustrative releasable conjugates of this type include:mPEG-O—(CH₂)_(b)—COOCH₂C(O)—NH-therapeutic peptide, andmPEG-O—(CH₂)_(b)—COO—CH(CH₃)—CH₂—C(O)—NH-therapeutic peptide, where thenumber of water-soluble polymers attached to therapeutic peptide can beanywhere from 1 to 4, or more preferably, from 1 to 3.

Carboxyl Coupling and Resulting Conjugates

Carboxyl groups represent another functional group that can serve as apoint of attachment to the therapeutic peptide. The conjugate will havethe following structure:

PEP-C(O)—X-POLY

where PEP-C(O)˜corresponds to a residue of a therapeutic peptide wherethe carbonyl is a carbonyl (derived from the carboxy group) of thetherapeutic peptide, X is a spacer moiety, such as a heteroatom selectedfrom O, N(H), and S, and POLY is a water-soluble polymer such as PEG,optionally terminating in an end-capping moiety.

The C(O)—X linkage results from the reaction between a polymericderivative bearing a terminal functional group and a carboxyl-containingtherapeutic peptide. As discussed above, the specific linkage willdepend on the type of functional group utilized. If the polymer isend-functionalized or “activated” with a hydroxyl group, the resultinglinkage will be a carboxylic acid ester and X will be O. If the polymerbackbone is functionalized with a thiol group, the resulting linkagewill be a thioester and X will be S. When certain multi-arm, branched orforked polymers are employed, the C(O)X moiety, and in particular the Xmoiety, may be relatively more complex and may include a longer linkerstructure.

Polymeric reagents containing a hydrazide moiety are also suitable forconjugation at a carbonyl. To the extent that the therapeutic peptidedoes not contain a carbonyl moiety, a carbonyl moiety can be introducedby reducing any carboxylic acid functionality (e.g., the C-terminalcarboxylic acid). Specific examples of polymeric reagents comprising ahydrazide moiety, along with the corresponding conjugates, are providedin Table 3, below. In addition, any polymeric reagent comprising anactivated ester (e.g., a succinimidyl group) can be converted to containa hydrazide moiety by reacting the polymer activated ester withhydrazine (NH₂—NH₂) or tert-butyl carbamate [NH₂NHCO₂C(CH₃)₃]. In thetable, the variable (n) represents the number of repeating monomericunits and “═C-(PEP)” represents a residue of a therapeutic peptidefollowing conjugation to the polymeric reagent were the underlined C ispart of the therapeutic peptide. Optionally, the hydrazone linkage canbe reduced using a suitable reducing agent. While each polymeric portion[e.g., (OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presented in Table 3 terminatesin a “CH₃” group, other groups (such as H and benzyl) can be substitutedtherefor.

TABLE 3 Carboxyl-Specific Polymeric Reagents and the GM-TherapeuticPeptide Conjugates Formed Therefrom Polymeric Reagent CorrespondingConjugate

Thiol Coupling and Resulting Conjugates

Thiol groups contained within the therapeutic peptide can serve aseffective sites of attachment for the water-soluble polymer. The thiolgroups contained in cysteine residues of the therapeutic peptide can bereacted with an activated PEG that is specific for reaction with thiolgroups, e.g., an N-maleimidyl polymer or other derivative, as describedin, for example, U.S. Pat. No. 5,739,208, WO 01/62827, and in Table 4below. In certain embodiments, cysteine residues may be introduced inthe therapeutic peptide and may be used to attach a water-solublepolymer.

Specific examples of the reagents themselves, along with thecorresponding conjugates, are provided in Table 4 below. In the table,the variable (n) represents the number of repeating monomeric units and“—S-(PEP)” represents a residue of a therapeutic peptide followingconjugation to the water-soluble polymer, where the S represents theresidue of a therapeutic peptide thiol group. While each polymericportion [e.g., (OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presented in Table 4terminates in a “CH₃” group, other end-capping groups (such as H andbenzyl) or reactive groups may be used as well.

TABLE 4 Thiol-Specific Polymeric Reagents and the Therapeutic peptideConjugates Formed Therefrom Polymeric Reagent Corresponding Conjugate

With respect to conjugates formed from water-soluble polymers bearingone or more maleimide functional groups (regardless of whether themaleimide reacts with an amine or thiol group on the therapeuticpeptide), the corresponding maleamic acid form(s) of the water-solublepolymer can also react with the therapeutic peptide. Under certainconditions (e.g., a pH of about 7-9 and in the presence of water), themaleimide ring will “open” to form the corresponding maleamic acid. Themaleamic acid, in turn, can react with an amine or thiol group of atherapeutic peptide. Exemplary maleamic acid-based reactions areschematically shown below. POLY represents the water-soluble polymer,and ˜S-PEP represents a residue of a therapeutic peptide, where the S isderived from a thiol group of the therapeutic peptide.

Thiol PEGylation is specific for free thiol groups on the therapeuticpeptide. Typically, a polymer maleimide is conjugated to asulfhydryl-containing therapeutic peptide at pHs ranging from about 6-9(e.g., at 6, 6.5, 7, 7.5, 8, 8.5, or 9), more preferably at pHs fromabout 7-9, and even more preferably at pHs from about 7 to 8. Generally,a slight molar excess of polymer maleimide is employed, for example, a1.5 to 15-fold molar excess, preferably a 2-fold to 10 fold molarexcess. Reaction times generally range from about 15 minutes to severalhours, e.g., 8 or more hours, at room temperature. For stericallyhindered sulfhydryl groups, required reaction times may be significantlylonger. Thiol-selective conjugation is preferably conducted at pHsaround 7. Temperatures for conjugation reactions are typically, althoughnot necessarily, in the range of from about 0° C. to about 40° C.;conjugation is often carried out at room temperature or less.Conjugation reactions are often carried out in a buffer such as aphosphate or acetate buffer or similar system.

With respect to reagent concentration, an excess of the polymericreagent is typically combined with the therapeutic peptide. Theconjugation reaction is allowed to proceed until substantially nofurther conjugation occurs, which can generally be determined bymonitoring the progress of the reaction over time.

Progress of the reaction can be monitored by withdrawing aliquots fromthe reaction mixture at various time points and analyzing the reactionmixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitableanalytical method. Once a plateau is reached with respect to the amountof conjugate formed or the amount of unconjugated polymer remaining, thereaction is assumed to be complete. Typically, the conjugation reactiontakes anywhere from minutes to several hours (e.g., from 5 minutes to 24hours or more). The resulting product mixture is preferably, but notnecessarily purified, to separate out excess reagents, unconjugatedreactants (e.g., therapeutic peptide) undesired multi-conjugatedspecies, and free or unreacted polymer. The resulting conjugates canthen be further characterized using analytical methods such as MALDI,capillary electrophoresis, gel electrophoresis, and/or chromatography.

An illustrative therapeutic peptide conjugate formed by reaction withone or more therapeutic peptide thiol groups may possess the followingstructure:

POLY-X_(0,1)—C(O)Z—Y—S—S-(PEP)

where POLY is a water-soluble polymer, X is an optional linker, Z is aheteroatom selected from the group consisting of O, NH, and S, and Y isselected from the group consisting of C₂₋₁₀ alkyl, C₂₋₁₀ substitutedalkyl, aryl, and substituted aryl, and —S-PEP is a residue of atherapeutic peptide, where the S represents the residue of a therapeuticpeptide thiol group. Such polymeric reagents suitable for reaction witha therapeutic peptide to result in this type of conjugate are describedin U.S. Patent Application Publication No. 2005/0014903, which isincorporated herein by reference.

With respect to polymeric reagents suitable for reacting with atherapeutic peptide thiol group, those described here and elsewhere canbe obtained from commercial sources. In addition, methods for preparingpolymeric reagents are described in the literature.

Additional Conjugates and Features Thereof

As is the case for any therapeutic peptide polymer conjugate of theinvention, the attachment between the therapeutic peptide andwater-soluble polymer can be direct, wherein no intervening atoms arelocated between the therapeutic peptide and the polymer, or indirect,wherein one or more atoms are located between the therapeutic peptideand polymer. With respect to the indirect attachment, a “spacer moietyor linker” serves as a link between the therapeutic peptide and thewater-soluble polymer. The one or more atoms making up the spacer moietycan include one or more of carbon atoms, nitrogen atoms, sulfur atoms,oxygen atoms, and combinations thereof. The spacer moiety can comprisean amide, secondary amine, carbamate, thioether, and/or disulfide group.Nonlimiting examples of specific spacer moieties (including “X”, X¹, X²,and X³) include those selected from the group consisting of —O—, —S—,—S—S—, —C(O)—, —C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—, —C(O)O—CH₂—,—OC(O)—CH₂—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]_(h)—(OCH2CH2)_(j)-, bivalent cycloalkyl group, —O—,—S—, an amino acid, —N(R⁶)—, and combinations of two or more of any ofthe foregoing, wherein R⁶ is H or an organic radical selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) iszero to six, and (j) is zero to 20. Other specific spacer moieties havethe following structures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and —O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, whereinthe subscript values following each methylene indicate the number ofmethylenes contained in the structure, e.g., (CH₂)₁₋₆ means that thestructure can contain 1, 2, 3, 4, 5 or 6 methylenes. Additionally, anyof the above spacer moieties may further include an ethylene oxideoligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e.,—(CH₂CH₂O)₁₋₂₀]. That is, the ethylene oxide oligomer chain can occurbefore or after the spacer moiety, and optionally in between any twoatoms of a spacer moiety comprised of two or more atoms. Also, theoligomer chain would not be considered part of the spacer moiety if theoligomer is adjacent to a polymer segment and merely represent anextension of the polymer segment.

As indicated above, in some instances the water-soluble polymer-(PEP)conjugate will include a non-linear water-soluble polymer. Such anon-linear water-soluble polymer encompasses a branched water-solublepolymer (although other non linear water-soluble polymers are alsocontemplated). Thus, in one or more embodiments of the invention, theconjugate comprises a therapeutic peptide covalently attached, eitherdirectly or through a spacer moiety comprised of one or more atoms, to abranched water-soluble polymer, at in a non-limiting example, aninternal or N-terminal amine. As used herein, an internal amine is anamine that is not part of the N-terminal amino acid (meaning not onlythe N-terminal amine, but any amine on the side chain of the N-terminalamino acid).

Although such conjugates include a branched water-soluble polymerattached (either directly or through a spacer moiety) to a therapeuticpeptide at an internal amino acid of the therapeutic peptide, additionalbranched water-soluble polymers can also be attached to the sametherapeutic peptide at other locations as well. Thus, for example, aconjugate including a branched water-soluble polymer attached (eitherdirectly or through a spacer moiety) to a therapeutic peptide at aninternal amino acid of the therapeutic peptide, can further include anadditional branched water-soluble polymer covalently attached, eitherdirectly or through a spacer moiety comprised of one or more atoms, tothe N-terminal amino acid residue, such as at the N-terminal amine.

One preferred branched water-soluble polymer comprises the followingstructure:

wherein each (n) is independently an integer having a value of from 3 to4000, or more preferably, from about 10 to 1800.

Also forming part of the invention are multi-armed polymer conjugatescomprising a polymer scaffold having 3 or more polymer arms eachsuitable for capable of covalent attachment of a therapeutic peptide.

Exemplary conjugates in accordance with this embodiment of the inventionwill generally comprise the following structure:

RPOLY-X-PEP)_(y)

wherein R is a core molecule as previously described, POLY is awater-soluble polymer, X is a cleavable, e.g., hydrolyzable linkage, andy ranges from about 3 to 15.

More particularly, such a conjugate may comprise the structure:

where m is selected from 3, 4, 5, 6, 7, and 8.

In yet a related embodiment, the therapeutic peptide conjugate maycorrespond to the structure:

where R is a core molecule as previously described, X is —NH—P—Z—C(O)Pis a spacer, Z is —O—, —NH—, or —CH₂—, —O-PEP is a hydroxyl residue of atherapeutic peptide, and y is 3 to 15. Preferably, X is a residue of anamino acid.

Purification

The therapeutic peptide polymer conjugates described herein can bepurified to obtain/isolate different conjugate species. Specifically, aproduct mixture can be purified to obtain an average of anywhere fromone, two, or three or even more PEGs per therapeutic peptide. In oneembodiment of the invention, preferred therapeutic peptide conjugatesare mono-conjugates. The strategy for purification of the finalconjugate reaction mixture will depend upon a number of factors,including, for example, the molecular weight of the polymeric reagentemployed, the therapeutic peptide, and the desired characteristics ofthe product—e.g., monomer, dimer, particular positional isomers, etc.

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography and/or ion exchangechromatography. Gel filtration chromatography may be used to fractionatedifferent therapeutic peptide conjugates (e.g., 1-mer, 2-mer, 3-mer, andso forth, wherein “1-mer” indicates one polymer molecule per therapeuticpeptide, “2-mer” indicates two polymers attached to therapeutic peptide,and so on) on the basis of their differing molecular weights (where thedifference corresponds essentially to the average molecular weight ofthe water-soluble polymer). While this approach can be used to separatePEG and other therapeutic peptide polymer conjugates having differentmolecular weights, this approach is generally ineffective for separatingpositional isomers having different polymer attachment sites within thetherapeutic peptide. For example, gel filtration chromatography can beused to separate from each other mixtures of PEG 1-mers, 2-mers, 3-mers,and so forth, although each of the recovered PEG-mer compositions maycontain PEGs attached to different reactive amino groups (e.g., lysineresidues) or other functional groups of the therapeutic peptide.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, (iii) iodine testing for PEG content (Sims et al. (1980) Anal.Biochem, 107:60-63), and (iv) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS PAGE), followed by staining with barium iodide.

Separation of positional isomers is typically carried out by reversephase chromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) orby ion exchange chromatography using an ion exchange column, e.g., aDEAE- or CM-Sepharose™ ion exchange column available from AmershamBiosciences. Either approach can be used to separate polymer-therapeuticpeptide isomers having the same molecular weight (positional isomers).

The resulting purified compositions are preferably substantially free ofthe non-conjugated therapeutic peptide. In addition, the compositionspreferably are substantially free of all other non-covalently attachedwater-soluble polymers.

Compositions Compositions of Conjugate Isomers

Also provided herein are compositions comprising any one or more of thetherapeutic peptide polymer conjugates described herein. In certaininstances, the composition will comprise a plurality of therapeuticpeptide polymer conjugates. For instance, such a composition maycomprise a mixture of therapeutic peptide polymer conjugates having one,two, three and/or even four water-soluble polymer molecules covalentlyattached to sites on the therapeutic peptide. That is to say, acomposition of the invention may comprise a mixture of monomer, dimer,and possibly even trimer or 4-mer. Alternatively, the composition maypossess only mono-conjugates, or only di-conjugates, etc. Amono-conjugate therapeutic peptide composition will typically comprisetherapeutic peptide moieties having only a single polymer covalentlyattached thereto, e.g., preferably releasably attached. A mono-conjugatecomposition may comprise only a single positional isomer, or maycomprise a mixture of different positional isomers having polymercovalently attached to different sites within the therapeutic peptide.

In yet another embodiment, a therapeutic peptide conjugate may possessmultiple therapeutic peptides covalently attached to a singlemulti-armed polymer having 3 or more polymer arms. Typically, thetherapeutic peptide moieties are each attached at the same therapeuticpeptide amino acid site, e.g., the N-terminus.

With respect to the conjugates in the composition, the composition willtypically satisfy one or more of the following characteristics: at leastabout 85% of the conjugates in the composition will have from one tofour polymers attached to the therapeutic peptide; at least about 85% ofthe conjugates in the composition will have from one to three polymersattached to the therapeutic peptide; at least about 85% of theconjugates in the composition will have from one to two polymersattached to the therapeutic peptide; or at least about 85% of theconjugates in the composition will have one polymer attached to thetherapeutic peptide (e.g., be monoPEGylated); at least about 95% of theconjugates in the composition will have from one to four polymersattached to the therapeutic peptide; at least about 95% of theconjugates in the composition will have from one to three polymersattached to the therapeutic peptide; at least about 95% of theconjugates in the composition will have from one to two polymersattached to the therapeutic peptide; at least about 95% of theconjugates in the composition will have one polymers attached to thetherapeutic peptide; at least about 99% of the conjugates in thecomposition will have from one to four polymers attached to thetherapeutic peptide; at least about 99% of the conjugates in thecomposition will have from one to three polymers attached to thetherapeutic peptide; at least about 99% of the conjugates in thecomposition will have from one to two polymers attached to thetherapeutic peptide; and at least about 99% of the conjugates in thecomposition will have one polymer attached to the therapeutic peptide(e.g., be monoPEGylated).

In one or more embodiments, the conjugate-containing composition is freeor substantially free of albumin.

In one or more embodiments of the invention, a pharmaceuticalcomposition is provided comprising a conjugate comprising a therapeuticpeptide covalently attached, e.g., releasably, to a water-solublepolymer, wherein the water-soluble polymer has a weight-averagemolecular weight of greater than about 2,000 Daltons; and apharmaceutically acceptable excipient.

Control of the desired number of polymers for covalent attachment totherapeutic peptide is achieved by selecting the proper polymericreagent, the ratio of polymeric reagent to the Therapeutic peptide,temperature, pH conditions, and other aspects of the conjugationreaction. In addition, reduction or elimination of the undesiredconjugates (e.g., those conjugates having four or more attachedpolymers) can be achieved through purification mean as previouslydescribed.

For example, the water-soluble polymer-(therapeutic peptide) conjugatescan be purified to obtain/isolate different conjugated species.Specifically, the product mixture can be purified to obtain an averageof anywhere from one, two, three, or four PEGs per therapeutic peptide,typically one, two or three PEGs per therapeutic peptide. In one or moreembodiments, the product comprises one PEG per therapeutic peptide,where PEG is releasably (via hydrolysis) attached to PEG polymer, e.g.,a branched or straight chain PEG polymer.

Pharmaceutical Compositions

Optionally, a therapeutic peptide conjugate composition of the inventionwill comprise, in addition to the therapeutic peptide conjugate, apharmaceutically acceptable excipient. More specifically, thecomposition may further comprise excipients, solvents, stabilizers,membrane penetration enhancers, etc., depending upon the particular modeof administration and dosage form.

Pharmaceutical compositions of the invention encompass all types offormulations and in particular those that are suited for injection,e.g., powders or lyophilates that can be reconstituted as well asliquids, as well as for inhalation. Examples of suitable diluents forreconstituting solid compositions prior to injection includebacteriostatic endotoxin-free water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof. With respect to liquidpharmaceutical compositions, solutions and suspensions are envisioned.

Exemplary pharmaceutically acceptable excipients include, withoutlimitation, carbohydrates, inorganic salts, antimicrobial agents,antioxidants, surfactants, buffers, acids, bases, and combinationsthereof.

Representative carbohydrates for use in the compositions of the presentinvention include sugars, derivatized sugars such as alditols, aldonicacids, esterified sugars, and sugar polymers. Exemplary carbohydrateexcipients suitable for use in the present invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like.Preferred, in particular for formulations intended for inhalation, arenon-reducing sugars, sugars that can form a substantially dry amorphousor glassy phase when combined with the composition of the presentinvention, and sugars possessing relatively high glass transitiontemperatures, or Tgs (e.g., Tgs greater than 40° C., or greater than 50°C., or greater than 60° C., or greater than 70° C., or having Tgs of 80°C. and above). Such excipients may be considered glass-formingexcipients.

Additional excipients include amino acids, peptides and particularlyoligomers comprising 2-9 amino acids, or 2-5 mers, and polypeptides, allof which may be homo or hetero species.

Exemplary protein excipients include albumins such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,hemoglobin, and the like. The compositions may also include a buffer ora pH-adjusting agent, typically but not necessarily a salt prepared froman organic acid or base. Representative buffers include organic acidsalts of citric acid, ascorbic acid, gluconic acid, carbonic acid,tartaric acid, succinic acid, acetic acid, or phthalic acid. Othersuitable buffers include Tris, tromethamine hydrochloride, borate,glycerol phosphate, and phosphate. Amino acids such as glycine are alsosuitable.

The compositions of the present invention may also include one or moreadditional polymeric excipients/additives, e.g., polyvinylpyrrolidones,derivatized celluloses such as hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose, FICOLLs (apolymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin.

The compositions may further include flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antioxidants, antistaticagents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN80,” and pluronics such as F68 and F88, available from BASF), sorbitanesters, lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, although preferably notin liposomal form), fatty acids and fatty esters, steroids (e.g.,cholesterol), and chelating agents (e.g., zinc and other such suitablecations). The use of certain di-substituted phosphatidylcholines forproducing perforated microstructures (i.e., hollow, porous microspheres)may also be employed.

Other pharmaceutical excipients and/or additives suitable for use in thecompositions according to the present invention are listed in“Remington: The Science & Practice of Pharmacy,” 21^(st) ed., Williams &Williams, (2005), and in the “Physician's Desk Reference,” 60th ed.,Medical Economics, Montvale, N.J. (2006).

The amount of the therapeutic peptide conjugate (i.e., the conjugateformed between the active agent and the polymeric reagent) in thecomposition will vary depending on a number of factors, but willoptimally be a therapeutically effective amount when the composition isstored in a unit dose container (e.g., a vial). In addition, apharmaceutical preparation, if in solution form, can be housed in asyringe. A therapeutically effective amount can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient or excipients will be present in thecomposition in an amount of about 1% to about 99% by weight, from about5% to about 98% by weight, from about 15 to about 95% by weight of theexcipient, or with concentrations less than 30% by weight. In general, ahigh concentration of the therapeutic peptide is desired in the finalpharmaceutical formulation.

Combination of Actives

A composition of the invention may also comprise a mixture ofwater-soluble polymer-(therapeutic peptide) conjugates and unconjugatedtherapeutic peptide, to thereby provide a mixture of fast-acting andlong-acting therapeutic peptide.

Additional pharmaceutical compositions in accordance with the inventioninclude those comprising, in addition to an extended-action therapeuticpeptide water-soluble polymer conjugate as described herein, a rapidacting therapeutic peptide polymer conjugate where the water-solublepolymer is releasably attached to a suitable location on the therapeuticpeptide.

Administration

The therapeutic peptide conjugates of the invention can be administeredby any of a number of routes including without limitation, oral, rectal,nasal, topical (including transdermal, aerosol, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenousand intradermal), intrathecal, and pulmonary. Preferred forms ofadministration include parenteral and pulmonary. Suitable formulationtypes for parenteral administration include ready-for-injectionsolutions, dry powders for combination with a solvent prior to use,suspensions ready for injection, dry insoluble compositions forcombination with a vehicle prior to use, and emulsions and liquidconcentrates for dilution prior to administration, among others.

In some embodiments of the invention, the compositions comprising thepeptide-polymer conjugates may further be incorporated into a suitabledelivery vehicle. Such delivery vehicles may provide controlled and/orcontinuous release of the conjugates and may also serve as a targetingmoiety. Non-limiting examples of delivery vehicles include, adjuvants,synthetic adjuvants, microcapsules, microparticles, liposomes, and yeastcell wall particles. Yeast cells walls may be variously processed toselectively remove protein component, glucan, or mannan layers, and arereferred to as whole glucan particles (WGP), yeast beta-glucan mannanparticles (YGMP), yeast glucan particles (YGP), \Rhodotorula yeast cellparticles (YCP). Yeast cells such as S. cerevisiae and Rhodotorula sp.are preferred; however, any yeast cell may be used. These yeast cellsexhibit different properties in terms of hydrodynamic volume and alsodiffer in the target organ where they may release their contents. Themethods of manufacture and characterization of these particles aredescribed in U.S. Pat. Nos. 5,741,495; 4,810,646; 4,992,540; 5,028,703;5,607,677, and US Patent Applications Nos. 2005/0281781, and2008/0044438.

In one or more embodiments of the invention, a method is provided, themethod comprising delivering a conjugate to a patient, the methodcomprising the step of administering to the patient a pharmaceuticalcomposition comprising a therapeutic peptide polymer conjugate asprovided herein. Administration can be effected by any of the routesherein described. The method may be used to treat a patient sufferingfrom a condition that is responsive to treatment with therapeuticpeptide by administering a therapeutically effective amount of thepharmaceutical composition.

As previously stated, the method of delivering a therapeutic peptidepolymer conjugate as provided herein may be used to treat a patienthaving a condition that can be remedied or prevented by administrationof therapeutic peptide.

Certain conjugates of the invention, e.g., releasable conjugates,include those effective to release the therapeutic peptide, e.g., byhydrolysis, over a period of several hours or even days (e.g., 2-7 days,2-6 days, 3-6 days, 3-4 days) when evaluated in a suitable in-vivomodel.

The actual dose of the therapeutic peptide conjugate to be administeredwill vary depending upon the age, weight, and general condition of thesubject as well as the severity of the condition being treated, thejudgment of the health care professional, and conjugate beingadministered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature. Generally, a conjugate of the invention will bedelivered such that plasma levels of a therapeutic peptide are within arange of about 0.5 picomoles/liter to about 500 picomoles/liter. Incertain embodiments the conjugate of the invention will be deliveredsuch that plasma leves of a therapeutic peptide are within a range ofabout 1 picomoles/liter to about 400 picomoles/liter, a range of about2.5 picomoles/liter to about 250 picomoles/liter, a range of about 5picomoles/liter to about 200 picomoles/liter, or a range of about 10picomoles/liter to about 100 picomoles/liter.

On a weight basis, a therapeutically effective dosage amount of atherapeutic peptide conjugate as described herein will range from about0.01 mg per day to about 1000 mg per day for an adult. For example,dosages may range from about 0.1 mg per day to about 100 mg per day, orfrom about 1.0 mg per day to about 10 mg/day. On an activity basis,corresponding doses based on international units of activity can becalculated by one of ordinary skill in the art.

The unit dosage of any given conjugate (again, such as provided as partof a pharmaceutical composition) can be administered in a variety ofdosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which arewithin the skill of the art. Such techniques are fully explained in theliterature. Reagents and materials are commercially available unlessspecifically stated to the contrary. See, for example, J. March,Advanced Organic Chemistry Reactions Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992), supra.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level.

Although other abbreviations known by one having ordinary skill in theart will be referenced, other reagents and materials will be used, andother methods known by one having ordinary skill in the art will beused, the following list and methods description is provided for thesake of convenience.

ABBREVIATIONS

-   mPEG-SPA mPEG-succinimidyl propionate-   mPEG-SBA mPEG-succinimidyl butanoate-   mPEG-SPC mPEG-succinimidyl phenyl carbonate-   mPEG-OPSS mPEG-orthopyridyl-disulfide-   mPEG-MAL mPEG-maleimide, CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂-MAL-   mPEG-SMB mPEG-succinimidyl α-methylbutanoate,    CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—CH(CH₃)—C(O)—O-succinimide-   mPEG-ButyrALD    H₃O—(CH₂CH₂O)_(n)—CH₂CH₂—O—C(O)—NH—(CH₂CH₂O)₄.CH₂CH₂CH₂C(O)H-   mPEG-PIP CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)-piperidin-4-one-   mPEG-CM CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—O—CH₂—C(O)—OH)-   anh. Anhydrous-   CV column volume-   Fmoc 9-fluorenylmethoxycarbonyl-   NaCNBH₃ sodium cyanoborohydride-   HCl hydrochloric acid-   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   NMR nuclear magnetic resonance-   DCC 1,3-dicyclohexylcarbodiimide-   DMF dimethylformamide-   DMSO dimethyl sulfoxide-   DI deionized-   MW molecular weight-   K or kDa kilodaltons-   SEC Size exclusion chromatography-   HPLC high performance liquid chromatography-   FPLC fast protein liquid chromatography-   SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis-   MALDI-TOF Matrix Assisted Laser Desorption Ionization Time-of-Flight-   TLC Thin Layer Chromatography-   THF Tetrahydrofuran

Materials

All PEG reagents referred to in the appended examples are commerciallyavailable unless otherwise indicated.

mPEG Reagent Preparation

Typically, a water-soluble polymer reagent is used in the preparation ofpeptide conjugates of the invention. For purposes of the presentinvention, a water-soluble polymer reagent is a water-solublepolymer-containing compound having at least one functional group thatcan react with a functional group on a peptide (e.g., the N-terminus,the C-terminus, a functional group associated with the side chain of anamino acid located within the peptide) to create a covalent bond. Takinginto account the known reactivity of the functional group(s) associatedwith the water-soluble polymer reagent, it is possible for one ofordinary skill in the art to determine whether a given water-solublepolymer reagent will form a covalent bond with the functional group(s)of a peptide.

Representative polymeric reagents and methods for conjugating suchpolymers to an active moiety are known in the art, and are, e.g.,described in Harris, J. M. and Zalipsky, S., eds, Poly(ethylene glycol),Chemistry and Biological Applications, ACS, Washington, 1997; Veronese,F., and J. M Harris, eds., Peptide and Protein PEGylation, Advanced DrugDelivery Reviews, 54(4); 453-609 (2002); Zalipsky, S., et al., “Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, ed., Plenus Press, New York (1992); Zalipsky(1995) Advanced Drug Reviews 16:157-182, and in Roberts, et al., Adv.Drug Delivery Reviews, 54, 459-476 (2002).

Additional PEG reagents suitable for use in forming a conjugate of theinvention, and methods of conjugation are described in ShearwaterCorporation, Catalog 2001; Shearwater Polymers, Inc., Catalogs, 2000 and1997-1998, and in Pasut. G., et al., Expert Opin. Ther. Patents (2004),14(5). PEG reagents suitable for use in the present invention alsoinclude those available from NOF Corporation (Tokyo, Japan), asdescribed generally on the NOF website (2006) under Products, HighPurity PEGs and Activated PEGs. Products listed therein and theirchemical structures are expressly incorporated herein by reference.Additional PEGs for use in forming a GLP-1 conjugate of the inventioninclude those available from Polypure (Norway) and from QuantaBioDesignLTD (Powell, Ohio), where the contents of their online catalogs (2006)with respect to available PEG reagents are expressly incorporated hereinby reference.

In addition, water-soluble polymer reagents useful for preparing peptideconjugates of the invention is prepared synthetically. Descriptions ofthe water-soluble polymer reagent synthesis can be found in, forexample, U.S. Pat. Nos. 5,252,714, 5,650,234, 5,739,208, 5,932,462,5,629,384, 5,672,662, 5,990,237, 6,448,369, 6,362,254, 6,495,659,6,413,507, 6,376,604, 6,348,558, 6,602,498, and 7,026,440.

Example 1 Peptide G-mPEG Conjugates

Peptide G is an amino acid synthetic peptide containing residues 161-189of the 40 kDa laminin binding domain of 67LR, which has been found toinhibit laminin-coated melanoma cells from attaching to endothelialcells that express the 67 kDa laminin receptor (Gastronovo et al., J.Biol. Chem. 1991, 266, 20440-6. The 20 amino acid sequence isIle-Pro-Cys-Asn-Asn-Lys-Gly-Ala-His-Ser-Val-Gly-Leu-Met-Trp-Trp-Met-Leu-Ala-Arg,has been proposed as potential new antimetastatic agent. (Gastronovo etal., Cancer Res. 1991, 51, 5672-8).

a) mPEG-N^(ter)-Peptide G Via mPEG-SPC

Peptide G is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of Peptide G, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Peptide G prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Peptide G conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Peptide G-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Peptide G, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protected PeptideG (Prot-Peptide G, e.g,Fmoc-Ile-Pro-Cys(tBu)-Asn-Asn-Lys(Fmoc)-Gly-Ala-His-Ser(Dmab)-Val-Gly-Leu-Met-Trp-Trp-Met-Leu-Ala-Arg(Tos))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Peptide G is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Peptide G-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Peptide G-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Peptide G-Cys(S-mPEG)

mPEG-Maleimide is obtained having a molecular weight of 5 kDa and havingthe basic structure shown below:

mPEG-MAL, 5 kDa

Peptide G, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Peptide G Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Peptide G solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 2 OTS102-mPEG Conjugates

OTS-102 is an angiogenesis inhibitor for cancer treatment consisting ofKDR169, the nine amino acid sequence starting at residue 169 of VEGFR2.KDR169 activates CD8-positive CTL's in an HLA-A2402 dependent manner.Augmented CTL exerts cytotoxicity to tumor-associated neovascularendothelial cells expressing KDR (VEGF receptor), and shows anti-tumoractivity (see, U.S. Patent Application No. 2006/216301 A1 andOncoTherapy Sciences, Inc web site,http://www.oncotherapy.co.jp/eng/rd/page3.html). KDR169 has thesequence, Arg-Phe-Val-Pro-Asp-Gly-Asn-Arg-Ile (RFVPDGNRI) (see, Seq. No.8, in US2006/216301A1).

a) mPEG-N^(ter)-OTS102 Via mPEG-SPC

The 9-aa KDR169 peptide is prepared and purified according to standardautomated peptide synthesis or recombinant techniques known to thoseskilled in the art. An illustrative polymeric reagent, mPEG-SPC reagent,is covalently attached to the N-terminus of KDR169, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of KDR169 prepared in phosphate buffered saline, PBS, pH 7.4is added and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-OTS102 conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) OTS102-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of KDR169, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protected KDR169(Prot-KDR169, e.g.,Fmoc-Arg(Tos)-Phe-Val-Pro-Asp(OBz)-Gly-Asn-Arg(Tos)-Ile-OH) is preparedand purified according to standard automated peptide synthesistechniques known to those skilled in the art. mPEG-NH₂ 20 kDa, stored at−20° C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-NH₂,PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-KDR169 is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-KDR169-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theOTS102-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) OTS102-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of KDR169, to provide a Asp-conjugate formof the peptide. For coupling to the Asp residue, a protected KDR169(Prot2-KDR169, e.g.,Fmoc-Arg(Tos)-Phe-Val-Pro-Asp(OBz)-Gly-Asn-Arg(Tos)-Ile-O(tBu)) isprepared and purified according to standard automated peptide synthesistechniques known to those skilled in the art. Deprotection of theAsp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate for subsequentcoupling (Prot3-KDR169, e.g.,Fmoc-Arg(Tos)-Phe-Val-Pro-Asp(OH)-Gly-Asn-Arg(Tos)-Ile-O(tBu)). mPEG-NH₂20 kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. A 5-fold molar excess ofmPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt3-KDR169 is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-KDR169-(Asp-O-mPEG) conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theOTS102-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

d) mPEG-N^(ter)-OTS102 Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock OTS102 solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 3 Angiocol™-mPEG Conjugates

Angiocol™ is a recombinant protein derived from the non-collagenousdomain (alpha-2) of type IV collagen, which has been shown inpreclinical studies to inhibit macrovascular endothelial cellproliferation (new blood vessel growth), as well as tumour growth, in invitro and in vivo models by targeting the assembly and organization ofthe vascular basal lamina. Angiocol™ has been proposed for the treatmentof retinal neovascularization (Coleman et al., Microcirculation 2004,11, 530).

a) mPEG-N^(ter)-Angiocol Via mPEG-SPC

Angiocol™ is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of Angiocol™, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Angiocol™ prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Angiocol conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Angiocol-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Angiocol™, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedAngiocol™ (Prot-Angiocol™) is prepared and purified according tostandard automated peptide synthesis techniques known to those skilledin the art. mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed toambient temperature. The reaction is performed at room temperature.About 3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-Angiocol™ isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Angiocol-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theAngiocol-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Angiocol™ Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Angiocol™ solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 4 ABT-510 (Antiangiogenic Peptide Group)-mPEG Conjugates

ABT-510 is nonapeptide analogue that mimics the anti-angiogenic activityof the endogenous protein thrombospondin-1 (TSP-1) which is indevelopment for treatment of advanced malignancies. ABT-510 blocks theactions of multiple pro-angiogenic growth factors known to play a rolein cancer related blood vessel growth, such as VEGF, bFGF, HGF, and IL-8(Haviv et al., J. Med. Chem. 2005, 48, 2838; Baker et al., J. Clin.Oncol. 2005, 23, 9013). In human studies, ABT-510 was found to be safeand have efficacy in phase I trials in combination regimens (Gietema etal., Ann. Oncol. 2006, 17, 1320-7).NAc-Sar-Gly-Val-(d-allo-Ile)-Thr-Nva-Ile-Arg-ProNEt (PubChem SubstanceID: 12015488)

a) mPEG-N^(ter)-ABT-510 Via mPEG-SPC

ABT-510 is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art,without the N-terminal acetyl group (NH₂-ABT-510). An illustrativepolymeric reagent, mPEG-SPC reagent, is covalently attached to theN-terminus of NH₂-ABT-510, to provide a N^(ter)-conjugate form of thepeptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, is warmed toambient temperature. The reaction is performed at room temperature.About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used basedupon absolute peptide content. The mPEG-SPC reagent is weighed into aglass vial containing a magnetic stirrer bar. A solution of NH₂-ABT-510prepared in phosphate buffered saline, PBS, pH 7.4 is added and themixture is stirred using a magnetic stirrer until the mPEG-SPC is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The reaction is optionallyquenched to terminate the reaction. The pH of the conjugate solution atthe end of the reaction is measured and further acidified by addition of0.1 M HCl, if necessary, to bring the pH of the final solution to about5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC(C18) to determine the extent of mPEG-N^(ter)-ABT-510 conjugateformation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) ABT-510-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of ABT-510, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedABT-510, lacking the C-terminal ethyl amide (Prot-ABT-510, e.g.,NAc-Sar(tBu)-Gly-Val-(d-allo-Ile)-Thr(tBu)-Nva-Ile-Arg(Tos)-Pro-OH) isprepared and purified according to standard automated peptide synthesistechniques known to those skilled in the art. mPEG-NH₂ 20 kDa, stored at−20° C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-NH₂,PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-ABT-510 is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-ABT-510-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theABT-510-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-ABT-510 Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock NH₂-ABT-510 (as in Example 4a) solution and mixed well. After theaddition of the mPEG-SMB, the pH of the reaction mixture is determinedand adjusted to 6.7 to 6.8 using conventional techniques. To allow forcoupling of the mPEG-SMB to the peptide via an amide linkage, thereaction solution is stirred for several hours (e.g., 5 hours) at roomtemperature in the dark or stirred overnight at 3-8° C. in a cold room,thereby resulting in a conjugate solution. The reaction is quenched witha 20-fold molar excess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 5 A6-mPEG Conjugates

A6 is a urokinase-derived eight amino-acid peptide,NAc-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-NH₂, with anti-angiogenic propertieswhich has been shown to suppres metastases and prolong the life span ofprostate tumor-bearing mice (Boyd et al., Am. J. Pathology 2003, 162.619).

a) mPEG-N^(ter)-A6 Via mPEG-SPC

A6 is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art,without the N-terminal acetyl group (NH₂-A6). An illustrative polymericreagent, mPEG-SPC reagent, is covalently attached to the N-terminus ofNH₂-A6, to provide a N^(ter)-conjugate form of the peptide. mPEG-SPC 20kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. About 3-5-fold molarexcess of mPEG-SPC 20 kDa reagent is used based upon absolute peptidecontent. The mPEG-SPC reagent is weighed into a glass vial containing amagnetic stirrer bar. A solution of NH₂-A6 prepared in phosphatebuffered saline, PBS, pH 7.4 is added and the mixture is stirred using amagnetic stirrer until the mPEG-SPC is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The reaction is optionally quenched to terminate thereaction. The pH of the conjugate solution at the end of the reaction ismeasured and further acidified by addition of 0.1M HCl, if necessary, tobring the pH of the final solution to about 5.5. The conjugate solutionis then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extentof mPEG-N^(ter)-A6 conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) A6-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of A6, to provide a C^(ter)-conjugate form ofthe peptide. For coupling to the C-terminus, a protected A6, lacking theC-terminal amide (Prot-A6, e.g.,NAc-Lys(Fmoc)-Pro-Ser(tBu)-Ser(tBu)-Pro-Pro-Glu(tBu)-Glu(tBu)-OH) isprepared and purified according to standard automated peptide synthesistechniques known to those skilled in the art. mPEG-NH₂ 20 kDa, stored at−20° C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-NH₂,PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-A6 is prepared in N,N-dimethylformamide is added and the mixture isstirred using a magnetic stirrer until the mPEG-NH₂ is fully dissolved.The stirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-A6-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theABT-510-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-A6 Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below: mPEG-SMB, 5 kDa, storedat −20° C. under argon, is warmed to ambient temperature. A five-foldexcess (relative to the amount of the peptide) of the warmed mPEG-SMB isdissolved in buffer to form a 10% reagent solution. The 10% reagentsolution is quickly added to the aliquot of a stock NH₂-A6 (as inExample 4a) solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) A6-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of A6, to provide a Glu-conjugate form ofthe peptide. For coupling to the Glu residue, a protected A6 (Prot2-A6,e.g.,NAc-Lys(Fmoc)-Pro-Ser(tBu)-Ser(tBu)-Pro-Pro-Glu(OBz)-Glu(tBu)-O(tBu)) isprepared and purified according to standard automated peptide synthesistechniques known to those skilled in the art. Deprotection of theGlu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate for subsequentcoupling (Prot3-A6, e.g.,NAc-Lys(Fmoc)-Pro-Ser(tBu)-Ser(tBu)-Pro-Pro-Glu-Glu(tBu)-O(tBu))mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-A6 is prepared inN,N-dimethylformamide is added and the mixture is stirred using amagnetic stirrer until the mPEG-NH₂ is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The conjugate solution is then analyzed by SDS-PAGEand RP-HPLC (C18) to determine the extent of Prot3-A6-(Glu-O-mPEG)conjugate formation. The remaining protecting groups are removed understandard deprotection conditions to yield the A6-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 6 Islet Neogenesis Gene Associated Protein (INGAP)-MpegConjugates

Islet Neogenesis-Associated Protein (INGAP) is a member of the Regfamily of proteins implicated in various settings of endogenouspancreatic regeneration. The expression of INGAP and other RegIIIproteins has also been linked with the induction of islet neogenesis inanimal models of disease and regeneration. Administration of a peptidefragment of INGAP (INGAP peptide) has been demonstrated to reversechemically induced diabetes as well as improve glycemic control andsurvival in an animal model of type 1 diabetes. (Lipsett et al., CellBiochem. Biophys. 2007, 48, 127). INGAP peptide (INGAPP) is a 15 aminoacid sequence contained within the 175 amino acid INGAP (see, aminoacids 103-117 of SEQ ID. NO: 2 of U.S. Pat. No. 5,834,590):Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser.

a) mPEG-N^(ter)-INGAPP Via mPEG-SPC

INGAPP is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of INGAPP, to provide a N^(ter)-conjugateform of the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof INGAPP prepared in phosphate buffered saline, PBS, pH 7.4 is addedand the mixture is stirred using a magnetic stirrer until the mPEG-SPCis fully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The reaction isoptionally quenched to terminate the reaction. The pH of the conjugatesolution at the end of the reaction is measured and further acidified byaddition of 0.1M HCl, if necessary, to bring the pH of the finalsolution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-INGAPP conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) INGAPP-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of INGAPP, to provide a C^(r)-conjugate formof the peptide. For coupling to the C-terminus, a protected INGAPP(Prot-INGAPP, e.gFmoc-Ile-Gly-Leu-His-Asp(tBu)-Pro-Ser(tBu)-His-Gly-Thr(tBu)-Leu-Pro-Asn-Gly-Ser(tBu)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-INGAPP is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-INGAPP-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theINGAPP-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-INGAPP Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of5,000 Daltons and having the basic structure shown below: mPEG-SMB, 5kDa, stored at −20° C. under argon, is warmed to ambient temperature. Afive-fold excess (relative to the amount of the peptide) of the warmedmPEG-SMB is dissolved in buffer to form a 10% reagent solution. The 10%reagent solution is quickly added to the aliquot of a stock INGAPPsolution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) INGAPP-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of INGAPP, to provide a Asp-conjugate formof the peptide. For coupling to the Asp residue, a protected INGAPP(Prot2-INGAPP, e.g.,Fmoc-Ile-Gly-Leu-His-Asp(OBz)-Pro-Ser(tBu)-His-Gly-Thr(tBu)-Leu-Pro-Asn-Gly-Ser(tBu)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-INGAPP, e.g.,Fmoc-Ile-Gly-Leu-His-Asp(OBz)-Pro-Ser(tBu)-His-Gly-Thr(tBu)-Leu-Pro-Asn-Gly-Ser(tBu)-O(tBu)).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-INGAPP isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-INGAPP-(Asp-O-mPEG) conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theINGAPP-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 7 Tendamistat-mPEG Conjugates

Tendamistat (HOE 467) is 74 residue alpha-amylase inactivator whicheffectively attenuates starch digestion (Meyer et al., S. Afr. Med. J.1984, 66, 222), having the sequence,Asp-Thr-Thr-Val-Ser-Glu-Pro-Ala-Pro-Ser-Cys-Val-Thr-Leu-Tyr-Gln-Ser-Tip-Arg-Tyr-Ser-Gln-Ala-Asp-Asp-Gly-Cys-Ala-Glu-Thr-Val-Thr-Val-Lys-Val-Val-Tyr-Glu-Asp-Asp-Thr-Glu-Gly-Leu-Cys-Tyr-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr-Thr-Val-Gly-Asp-Gly-Tyr-Ile-Gly-Ser-His-Gly-His-Ala-Arg-Tyr-Leu-Ala-Arg-Cys-Leu(DTTVSEPAPS CVTLYQSWRY SQADNGCAET VTVKVVYEDD TEGLCYAVAP GQITTVGDGYIGSHGHARYL ARCL) (PubChem Protein Accession No. CAA00655)

a) mPEG-N^(tre)-Tendamistat—Via mPEG-SPC

Tendamistat is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Tendamistat, to provide aN^(tre)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Tendamistat prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Tendamistat conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Tendamistat-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Tendamistat, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Tendamistat (Prot-Tendamistat, e.g.,Fmoc-Asp(tBu)-Thr(tBu)-Thr(tBu)-Val-Ser(tBu)-Glu(tBu)-Pro-Ala-Pro-Ser(tBu)-Cys(tBu)-Val-Thr(tBu)-Leu-Tyr(tBu)-Gln-Ser(tBu)-Trp-Arg(Tos)-Tyr-Ser(tBu)-Gln-Ala-Asp(tBu)-Asp(tBu)-Gly-Cys(tBu)-Ala-Glu(tBu)-Thr(tBu)-Val-Thr(tBu)-Val-Lys(Fmoc)-Val-Val-Tyr(tBu)-Glu(tBu)-Asp(tBu)-Asp(tBu)-Thr(tBu)-Glu(tBu)-Gly-Leu-Cys(tBu)-Tyr(tBu)-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr(tBu)-Thr(tBu)-Val-Gly-Asp(tBu)-Gly-Tyr(tBu)-Ile-Gly-Ser(tBu)-His-Gly-His-Ala-Arg(Tos)-Tyr(tBu)-Leu-Ala-Arg(Tos)-Cys(tBu)-Leu)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Tendamistat is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Tendamistat-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Tendamistat-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Tendamistat-Cys(S-mPEG)

Tendamistat, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Tendamistat Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Tendamistat solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Tendamistat-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of Tendamistat, to provide a Glu-conjugateform of the peptide. For coupling to the Glu residue, a protectedTendamistat (Prot2-Tendamistat, e.g,Fmoc-Asp(tBu)-Thr(tBu)-Thr(tBu)-Val-Ser(tBu)-Glu(OBz)-Pro-Ala-Pro-Ser(tBu)-Cys(tBu)-Val-Thr(tBu)-Leu-Tyr(tBu)-Gln-Ser(tBu)-Trp-Arg(Tos)-Tyr-Ser(tBu)-Gln-Ala-Asp(tBu)-Asp(tBu)-Gly-Cys(tBu)-Ala-Glu(tBu)-Thr(tBu)-Val-Thr(tBu)-Val-Lys(Fmoc)-Val-Val-Tyr(tBu)-Glu(tBu)-Asp(tBu)-Asp(tBu)-Thr(tBu)-Glu(tBu)-Gly-Leu-Cys(tBu)-Tyr(tBu)-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr(tBu)-Thr(tBu)-Val-Gly-Asp(tBu)-Gly-Tyr(tBu)-Ile-Gly-Ser(tBu)-His-Gly-His-Ala-Arg(Tos)-Tyr(tBu)-Leu-Ala-Arg(Tos)-Cys(tBu)-Leu(OtBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Glu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate forsubsequent coupling (Prot3-Tendamistat, e.g.,Fmoc-Asp(tBu)-Thr(tBu)-Thr(tBu)-Val-Ser(tBu)-Glu-Pro-Ala-Pro-Ser(tBu)-Cys(tBu)-Val-Thr(tBu)-Leu-Tyr(tBu)-Gln-Ser(tBu)-Trp-Arg(Tos)-Tyr-Ser(tBu)-Gln-Ala-Asp(tBu)-Asp(tBu)-Gly-Cys(tBu)-Ala-Glu(tBu)-Thr(tBu)-Val-Thr(tBu)-Val-Lys(Fmoc)-Val-Val-Tyr(tBu)-Glu(tBu)-Asp(tBu)-Asp(tBu)-Thr(tBu)-Glu(tBu)-Gly-Leu-Cys(tBu)-Tyr(tBu)-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr(tBu)-Thr(tBu)-Val-Gly-Asp(tBu)-Gly-Tyr(tBu)-Ile-Gly-Ser(tBu)-His-Gly-His-Ala-Arg(Tos)-Tyr(tBu)-Leu-Ala-Arg(Tos)-Cys(tBu)-Leu(OtBu))mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Tendamistat isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Tendamistat-(Glu-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Tendamistat-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 8 Recombinant Human Carperitide-mPEG conjugates

Carperitide (α-atriopeptin) is secreted by the heart, is a member of thenatriuretic peptide family which is comprised of peptides secreted byvarious organs. Carperitide is has been proposed for the treatment ofacute heart failure and shown therapeutic potential to treat peripheralarterial diseases refractory to conventional therapies (Park et al.,Endocrinology 2008, 149, 483). Carperitide has the amino acid sequenceSer-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr(SLRRSSCFGGRMDRIGAQSGLGCNSFRY).

a) mPEG-N^(ter)-Carperitide—Via mPEG-SPC

Carperitide can be prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Carperitide, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used, based upon absolute peptide content. ThemPEG-SPC reagent is weighed into a glass vial containing a magneticstirrer bar. A solution of Carperitide prepared in phosphate bufferedsaline, PBS, pH 7.4 is added and the mixture is rapidly stirred using amagnetic stirrer until the mPEG-SPC is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The reaction is optionally quenched to terminate thereaction. The pH of the conjugate solution at the end of the reaction ismeasured and further acidified by addition of 0.1M HCl, if necessary, tobring the pH of the final solution to about 5.5. The conjugate solutionis then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extentof mPEG-N^(ter)-Carperitide conjugate formation.

Using this same approach, other conjugates can be prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Carperitide-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Carperitide, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Carperitide (Prot-Carperitide, e.g.,Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(tBu)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-OH)can be prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. About3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-Carperitide isprepared in N,N-dimethylformamide is added and the mixture is rapidlystirred using a magnetic stirrer until the mPEG-NH₂ is fully dissolved.The stirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Carperitide-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Carperitide-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates can be prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Carperitide-Cys(S-mPEG)

Carperitide, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates can be prepared usingmPEG-MAL having other weight average molecular weights.

d) mPEG-N^(ter)-Carperitide Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Carperitide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates can be prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Carperitide-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of Carperitide, to provide a Asp-conjugateform of the peptide. For coupling to the Asp residue, a protectedCarperitide (Prot2-Carperitide, e.g.,Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(OBz)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-Carperitide, e.g.,Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-O(tBu)).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Carperitide isprepared in N,N-dimethylformamide is added and the mixture is rapidlystirred using a magnetic stirrer until the mPEG-NH₂ is fully dissolved.The stirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Carperitide-(Asp-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Carperitide-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates is prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 9 Urodilatin-mPEG Conjugates

Urodilatin is a member of the natriuretic peptide family which iscomprised of peptides secreted by various organs, has been studied foruse in treating various conditions, including renal failure orcongestive heart failure (see, e.g., U.S. Pat. Nos. 5,571,789 and6,831,064; Kentsch et al., Eur. J. Clin. Invest. 1992, 22, 662; Kentschet al., Eur. J. Clin. Invest. 1995, 25, 281; Elsner et al., Am. Heart J.1995, 129, 766; Forssmann et al., Clinical Pharmacology and Therapeutics1998, 64, 322; and US Patent Application Publication No.2006/0264376A1). Urodilatin has the amino acid sequence set forth inGenBank Accession No. 1506430A;Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr(TAPRSLRRSS CFGGRMDRIG AQSGLGCNSF RY). Urodilatin is also the 95-126fragment [ANP(95-126)] of atrial natriuretic peptide (ANP).

a) mPEG-N^(ter)-Urodilatin—Via mPEG-SPC

Urodilatin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Urodilatin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Urodilatin prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Urodilatin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Urodilatin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Urodilatin, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedUrodilatin (Prot-Urodilatin, e.g.,Fmoc-Thr(tBu)-Ala-Pro-Arg(Tos)-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(tBu)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Urodilatin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Urodilatin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Urodilatin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Urodilatin-Cys(S-mPEG)

Urodilatin, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Urodilatin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Urodilatin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Urodilatin-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of Urodilatin, to provide a Asp-conjugateform of the peptide. For coupling to the Asp residue, a protectedUrodilatin (Prot2-Urodilatin, e.g.,Fmoc-Thr(tBu)-Ala-Pro-Arg(Tos)-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(OBz)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-NH₂)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H2/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-Urodilatin, e.g.Fmoc-Thr(tBu)-Ala-Pro-Arg(Tos)-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-NH₂).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Urodilatin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Urodilatin-(Asp-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Urodilatin-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 10 Desirudin-mPEG Conjugates

Desirudin, a recombinant hirudin, is a member of a class ofanticoagulants that act by directly inhibiting thrombin. Desirudin actsvia a bivalent binding arrangement with both the active site andfibrinogen-binding site (exosite 1) of thrombin, and has been shown tobe useful in the prevention and management of thromboembolic disease,reducing the incidence of deep vein thrombosis (DVT) in patientsundergoing elective hip replacement, preventing restenosis aftercoronary angioplasty for unstable angina, and in the treatment of acutecoronary syndromes for patients in whom heparin therapy is not a viableoption (Matheson and Goa, Drugs 2000, 60, 679). Desirudin has theprimary sequenceVal-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.

a) mPEG-N^(tre)-Desirudin Via mPEG-SPC

Desirudin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of Desirudin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Desirudin prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Desirudin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Desirudin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Desirudin, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedDesirudin(Prot-Val-Val-Tyr(tBu)-Thr(tBu)-Asp(tBu)-Cys(tBu)-Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-Gln-Asn-Leu-Cys(tBu)-Leu-Cys-Glu(tBu)-Gly-Ser(tBu)-Asn-Val-Cys(tBu)-Gly-Gln-Gly-Asn-Lys(Fmoc)-Cys(tBu)-Ile-Leu-Gly-Ser(tBu)-Asp(tBu)-Gly-Glu(tBu)-Lys(Fmoc)-Asn-Gln-Cys(tBu)-Val-Thr(tBu)-Gly-Glu(tBu)-Gly-Thr(tBu)-Pro-Lys(Fmoc)-Pro-Gln-Ser(tBu)-His-Asn-Asp(tBu)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-Ile-Pro-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Leu-Gln-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Desirudin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Desirudin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Desirudin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Desirudin-Cys(S-mPEG)

Desirudin, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Desirudin Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Desirudin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Desirudin-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of Desirudin, to provide a Asp-conjugateform of the peptide. For coupling to the Asp residue, a protectedDesirudin (Prot2-Desirudin, e.g.Fmoc-Val-Val-Tyr(tBu)-Thr(tBu)-Asp(OBz)-Cys(tBu)-Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-Gln-Asn-Leu-Cys(tBu)-Leu-Cys-Glu(tBu)-Gly-Ser(tBu)-Asn-Val-Cys(tBu)-Gly-Gln-Gly-Asn-Lys(Fmoc)-Cys(tBu)-Ile-Leu-Gly-Ser(tBu)-Asp(tBu)-Gly-Glu(tBu)-Lys(Fmoc)-Asn-Gln-Cys(tBu)-Val-Thr(tBu)-Gly-Glu(tBu)-Gly-Thr(tBu)-Pro-Lys(Fmoc)-Pro-Gln-Ser(tBu)-His-Asn-Asp(tBu)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-Ile-Pro-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Leu-Gln-NH₂)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-Desirudin, e.g.Fmoc-Val-Val-Tyr(tBu)-Thr(tBu)-Asp-Cys(tBu)-Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-Gln-Asn-Leu-Cys(tBu)-Leu-Cys-Glu(tBu)-Gly-Ser(tBu)-Asn-Val-Cys(tBu)-Gly-Gln-Gly-Asn-Lys(Fmoc)-Cys(tBu)-Ile-Leu-Gly-Ser(tBu)-Asp(tBu)-Gly-Glu(tBu)-Lys(Fmoc)-Asn-Gln-Cys(tBu)-Val-Thr(tBu)-Gly-Glu(tBu)-Gly-Thr(tBu)-Pro-Lys(Fmoc)-Pro-Gln-Ser(tBu)-His-Asn-Asp(tBu)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-Ile-Pro-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Leu-Gln-NH₂).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Desirudin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Desirudin-(Asp-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Desirudin-Asp(O-mPEG) conjugate.

Example 11 Obestatin-mPEG Conjugates

Obestatin is 28-amino acid, acylated, orexigenic peptide that is aligand for growth hormone secretagogue receptors and is encoded by thesame gene that also encodes ghrelin, a peptide hormone that increasesappetite. Treatment of rats with obestatin suppressed food intake,inhibited jejunal contraction, and decreased body-weight gain (Zhang etal., Science 2005, 310, 996). Synthetic human obestatin is availablefrom California Peptide Research, Inc (Napa, Calif.), having thesequence,Phe-Asn-Ala-Pro-Phe-Asp-Val-Gly-Ile-Lys-Leu-Ser-Gly-Val-Gln-Tyr-Gln-Gln-His-Ser-Gln-Ala-Leu-NH₂(PubChem Substance ID: 47205412).

a) mPEG-IV-Obestatin Via mPEG-SPC

An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of Obestatin, to provide a N^(ter)-conjugateform of the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof Obestatin prepared in phosphate buffered saline, PBS, pH 7.4 is addedand the mixture is stirred using a magnetic stirrer until the mPEG-SPCis fully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The reaction isoptionally quenched to terminate the reaction. The pH of the conjugatesolution at the end of the reaction is measured and further acidified byaddition of 0.1M HCl, if necessary, to bring the pH of the finalsolution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-Mer-Obestatinconjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Obestatin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Obestatin, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedObestatin lacking the C-terminus amide (Prot-Obestatin, e.g.,Fmoc-Phe-Asn-Ala-Pro-Phe-Asp(tBu)-Val-Gly-Ile-Lys(Fmoc)-Leu-Ser(tBu)-Gly-Val-Gln-Tyr(tBu)-Gln-Gln-His-Ser(tBu)-Gln-Ala-Leu-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Obestatin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Obestatin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Obestatin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Obestatin-Cys(S-mPEG)

Obestatin, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Obestatin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Obestatin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Obestatin-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Obestatin (e.g.,Fmoc-Phe-Asn-Ala-Pro-Phe-Asp(tBu)-Val-Gly-Ile-Lys-Leu-Ser(tBu)-Gly-Val-Gln-Tyr(tBu)-Gln-Gln-His-Ser(tBu)-Gln-Ala-Leu-NH₂)solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theObestatin-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 12 ITF-1697(krocaptide)-mPEG Conjugates

ITF-1697 is a tetrapeptide, Gly-(N-Et)Lys-Pro-Arg (PubChem Compound ID:216295), which reduces mortality and tissue damage in lipopolysaccharide(LPS)-induced systemic endotoxemia and coronary ischemia andischemia/reperfusion (see, International Patent Application PublicationWO 1995/10531.). A randomized, double-blind study in patients with acutemyocardial infarction undergoing coronary revascularisation demonstratedreduce infarct size by radionuclide imaging (Syeda et al., Drugs R & D2004, 5, 141).

a) mPEG-N^(ter)-ITF-1697—Via mPEG-SPC

ITF-1697 is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of ITF-1697, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of ITF-1697 prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-ITF-1697 conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) IT F-1697-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of ITF-1697, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedITF-1697 (Prot-ITF-1697, e.g., Fmoc-Gly-(N-Et)Lys(Fmoc)-Pro-Arg(Tos)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-ITF-1697 is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-ITF-1697-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theITF-1697-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-ITF-1697 Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock ITF-1697 solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) ITF-1697-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected ITF-1697 (e.g., Fmoc-Gly-(N-Et)Lys-Pro-Arg(Tos)-O(tBu))solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theITF-1697-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 13 Oxyntomodulin-mPEG Conjugates

Oxyntomodulin (Amylin) is a 37-amino acid peptide derived fromproglucagon found in the colon, produced by the oxyntic (fundic) cellsof the oxyntic mucosa and is known to bind both the Glucagon-likepeptide-1 (GLP-1) and the glucagon receptors. A randomized,double-blind, placebo-controlled, cross-over study in humans has shownOxyntomodulin suppresses appetite and food intake (Cohen et al., J.Clin. Endocrin. Met. 2003, 88, 4696). Oxyntomodulin is commerciallyavailable from GenScript Corporation (Piscataway, N.J.; Cat. No.RP11278) with the sequence,Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH₂(KCNTATCATQ RLANFLVHSS NNFGAILSST NVGSNTY-NH₂).

a) mPEG-N^(ter)-Oxyntomodulin—Via mPEG-SPC

An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of Oxyntomodulin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Oxyntomodulin prepared in phosphate buffered saline, PBS,pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1M HCl, if necessary, to bring the pHof the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Oxyntomodulin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Oxyntomodulin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Oxyntomodulin, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Oxyntomodulin lacking the C-terminus amide(Prot-Oxyntomodulin, e.g.,Fmoc-Lys(Fmoc)-Cys(tBu)-Asn-Thr(tBu)-Ala-Thr(tBu)-Cys(tBu)-Ala-Thr(tBu)-Gln-Arg(Tos)-Leu-Ala-Asn-Phe-Leu-Val-His-Ser(tBu)-Ser(tBu)-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser(tBu)-Ser(tBu)-Thr(tBu)-Asn-Val-Gly-Ser(tBu)-Asn-Thr(tBu)-Tyr(tBu)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Oxyntomodulin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Oxyntomodulin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Oxyntomodulin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Oxyntomodulin-Cys(S-mPEG)

Oxyntomodulin, which has a thiol-containing cysteine residue, isdissolved in buffer. To this peptide solution is added a 3-5 fold molarexcess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperatureunder an inert atmosphere for several hours. Analysis of the reactionmixture reveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Oxyntomodulin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Oxyntomodulin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Oxyntomodulin-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Oxyntomodulin (e.g.,Fmoc-Lys-Cys(tBu)-Asn-Thr(tBu)-Ala-Thr(tBu)-Cys(tBu)-Ala-Thr(tBu)-Gln-Arg(Tos)-Leu-Ala-Asn-Phe-Leu-Val-His-Ser(tBu)-Ser(tBu)-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser(tBu)-Ser(tBu)-Thr(tBu)-Asn-Val-Gly-Ser(tBu)-Asn-Thr(tBu)-Tyr(tBu)-O(tBu))solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theOxyntomodulin-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 14 Cholecystokinin-mPEG Conjugates

Cholecystokinin is a peptide hormone secreted by the upper intestinalmucosa which increases gallbladder contraction, release of pancreaticexocrine (or digestive) enzymes, and is responsible for stimulating thedigestion of fat and proteins. Cholecystokinin has also been shown to bea physiologic regulator of gastric emptying in humans (Liddle et al., J.Clin. Invest. 1986, 77, 992). Cholecystokinin has the sequence,Met-Asn-Ser-Gly-Val-Cys-Leu-Cys-Val-Leu-Met-Ala-Val-Leu-Ala-Ala-Gly-Ala-Leu-Thr-Gln-Pro-Val-Pro-Pro-Ala-Asp-Pro-Ala-Gly-Ser-Gly-Leu-Gln-Arg-Ala-Glu-Glu-Ala-Pro-Arg-Arg-Gln-Leu-Arg-Val-Ser-Gln-Arg-Thr-Asp-Gly-Glu-Ser-Arg-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg-Tyr-Ile-Gln-Gln-Ala-Arg-Lys-Ala-Pro-Ser-Gly-Arg-Met-Ser-Ile-Val-Lys-Asn-Leu-Gln-Asn-Leu-Asp-Pro-Ser-His-Arg-Ile-Ser-Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg-Ser-Ala-Glu-Glu-Tyr-Glu-Tyr-Pro-Ser(MNSGVCLCVL MAVLAAGALT QPVPPADPAG SGLQRAEEAP RRQLRVSQRT DGESRAHLGALLARYIQQAR KAPSGRMSIV KNLQNLDPSH RISDRDYMGW MDFGRRSAEE YEYPS; PubChemProtein Accession No. AAA53094; Takahashi et al., Gene, 1986, 50, 353).

a) mPEG-N^(ter)-Cholecystokinin—Via mPEG-SPC

Cholecystokinin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Cholecystokinin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Cholecystokinin prepared in phosphate buffered saline,PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1M HCl, if necessary, to bring the pHof the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Cholecystokinin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Cholecystokinin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Cholecystokinin, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Cholecystokinin (Prot-Cholecystokinin, e.g.,Fmoc-Met-Asn-Ser(tBu)-Gly-Val-Cys(tBu)-Leu-Cys(tBu)-Val-Leu-Met-Ala-Val-Leu-Ala-Ala-Gly-Ala-Leu-Thr(tBu)-Gln-Pro-Val-Pro-Pro-Ala-Asp(tBu)-Pro-Ala-Gly-Ser(tBu)-Gly-Leu-Gln-Arg(Tos)-Ala-Glu(tBu)-Glu(tBu)-Ala-Pro-Arg(Tos)-Arg(Tos)-Gln-Leu-Arg(Tos)-Val-Ser(tBu)-Gln-Arg(Tos)-Thr(tBu)-Asp(tBu)-Gly-Glu(tBu)-Ser(tBu)-Arg(Tos)-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg(Tos)-Tyr(tBu)-Ile-Gln-Gln-Ala-Arg(Tos)-Lys(Fmoc)-Ala-Pro-Ser(tBu)-Gly-Arg(Tos)-Met-Ser(tBu)-Ile-Val-Lys(Fmoc)-Asn-Leu-Gln-Asn-Leu-Asp(tBu)-Pro-Ser(tBu)-His-Arg(Tos)-Ile-Ser(tBu)-Asp(tBu)-Arg(Tos)-Asp(tBu)-Tyr(tBu)-Met-Gly-Trp-Met-Asp(tBu)-Phe-Gly-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ala-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Glu(tBu)-Tyr(tBu)-Pro-Ser(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Cholecystokinin is prepared in N,N-dimethylformamide is added andthe mixture is stirred using a magnetic stirrer until the mPEG-NH₂ isfully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The conjugatesolution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine theextent of Prot-Cholecystokinin-C^(ter)-mPEG conjugate formation. Theremaining protecting groups are removed under standard deprotectionconditions to yield the Cholecystokinin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Cholecystokinin-Cys(S-mPEG)

Cholecystokinin, which has a thiol-containing cysteine residue, isdissolved in buffer. To this peptide solution is added a 3-5 fold molarexcess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperatureunder an inert atmosphere for several hours. Analysis of the reactionmixture reveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Cholecystokinin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Cholecystokinin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Cholecystokinin-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of Cholecystokinin, to provide aGlu-conjugate form of the peptide. For coupling to the Glu residue, aprotected Cholecystokinin (Prot2-Cholecystokinin, e.g.,Fmoc-Met-Asn-Ser(tBu)-Gly-Val-Cys(tBu)-Leu-Cys(tBu)-Val-Leu-Met-Ala-Val-Leu-Ala-Ala-Gly-Ala-Leu-Thr(tBu)-Gln-Pro-Val-Pro-Pro-Ala-Asp(tBu)-Pro-Ala-Gly-Ser(tBu)-Gly-Leu-Gln-Arg(Tos)-Ala-Glu(OBz)-Glu(tBu)-Ala-Pro-Arg(Tos)-Arg(Tos)-Gln-Leu-Arg(Tos)-Val-Ser(tBu)-Gln-Arg(Tos)-Thr(tBu)-Asp(tBu)-Gly-Glu(tBu)-Ser(tBu)-Arg(Tos)-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg(Tos)-Tyr(tBu)-Ile-Gln-Gln-Ala-Arg(Tos)-Lys(Fmoc)-Ala-Pro-Ser(tBu)-Gly-Arg(Tos)-Met-Ser(tBu)-Ile-Val-Lys(Fmoc)-Asn-Leu-Gln-Asn-Leu-Asp(tBu)-Pro-Ser(tBu)-His-Arg(Tos)-Ile-Ser(tBu)-Asp(tBu)-Arg(Tos)-Asp(tBu)-Tyr(tBu)-Met-Gly-Trp-Met-Asp(tBu)-Phe-Gly-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ala-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Glu(tBu)-Tyr(tBu)-Pro-Ser(tBu)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Glu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate forsubsequent coupling (Prot3-Cholecystokinin, e.g.,Fmoc-Met-Asn-Ser(tBu)-Gly-Val-Cys(tBu)-Leu-Cys(tBu)-Val-Leu-Met-Ala-Val-Leu-Ala-Ala-Gly-Ala-Leu-Thr(tBu)-Gln-Pro-Val-Pro-Pro-Ala-Asp(tBu)-Pro-Ala-Gly-Ser(tBu)-Gly-Leu-Gln-Arg(Tos)-Ala-Glu-Glu(tBu)-Ala-Pro-Arg(Tos)-Arg(Tos)-Gln-Leu-Arg(Tos)-Val-Ser(tBu)-Gln-Arg(Tos)-Thr(tBu)-Asp(tBu)-Gly-Glu(tBu)-Ser(tBu)-Arg(Tos)-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg(Tos)-Tyr(tBu)-Ile-Gln-Gln-Ala-Arg(Tos)-Lys(Fmoc)-Ala-Pro-Ser(tBu)-Gly-Arg(Tos)-Met-Ser(tBu)-Ile-Val-Lys(Fmoc)-Asn-Leu-Gln-Asn-Leu-Asp(tBu)-Pro-Ser(tBu)-His-Arg(Tos)-Ile-Ser(tBu)-Asp(tBu)-Arg(Tos)-Asp(tBu)-Tyr(tBu)-Met-Gly-Trp-Met-Asp(tBu)-Phe-Gly-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ala-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Glu(tBu)-Tyr(tBu)-Pro-Ser(tBu)-O(tBu))mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Cholecystokininis prepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Cholecystokinin-(Glu-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Cholecystokinin-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 15 Bactericidal Permeability Increasing (BPI) Protein-mPEGConjugates

Bactericidal permeability increasing protein (BPI) is a 487 residue (˜50kDa) protein which is part of the innate immune system and whichdisplays selective cytotoxicity toward gram-negative bacteria throughbinding to lipopolysaccharides produced by the bacteria. BPI has thesequence, MRENMARGPC NAPRWVSLMV LVAIGTAVTA AVNPGVVVRI SQKGLDYASQQGTAALQKEL KRIKIPDYSD SFKIKHLGKG HYSFYSMDIR EFQLPSSQIS MVPNVGLKFSISNANIKISG KWKAQKRFLK MSGNFDLSIE GMSISADLKL GSNPTSGKPT ITCSSCSSHINSVHVHISKS KVGWLIQLFH KKIESALRNK MNSQVCEKVT NSVSSKLQPY FQTLPVMTKIDSVAGINYGL VAPPATTAET LDVQMKGEFY SENHHNPPPF APPVMEFPAA HDRMVYLGLSDYFFNTAGLV YQEAGVLKMT LRDDMIPKES KFRLTTKFFG TFLPEVAKKF PNMKIQIHVSASTPPHLSVQ PTGLTFYPAV DVQAFAVLPN SSLASLFLIG MHTTGSMEVS AESNRLVGELKLDRLLLELK HSNIGPFPVE LLQDIMNYIV PILVLPRVNE KLQKGFPLPT PARVQLYNVVLQPHQNFLLF GADVVYK (PubChem Protein Accession No. AAA51841; Gray et al.,J. Biol. Chem. 1989, 264, 9505).

a) mPEG-N^(ter)-BPI—Via mPEG-SPC

BPI is prepared and purified according to standard recombinanttechniques known to those skilled in the art. An illustrative polymericreagent, mPEG-SPC reagent, is covalently attached to the N-terminus ofBPI, to provide a M″-conjugate form of the peptide. mPEG-SPC 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.The mPEG-SPC reagent is weighed into a glass vial containing a magneticstirrer bar. A solution of BPI prepared in phosphate buffered saline,PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1M HCl, if necessary, to bring the pHof the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-BPI conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) BPI-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of BPI, to provide a C^(ter)-conjugate formof the peptide. mPEG-NH₂ 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of BPI is prepared inN,N-dimethylformamide is added and the mixture is stirred using amagnetic stirrer until the mPEG-NH₂ is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The conjugate solution is then analyzed by SDS-PAGEand RP-HPLC (C18) to determine the extent of BPI-C^(ter)-mPEG conjugateformation. The C^(ter) conjugate is isolated and purified according thegeneral procedure outlined above.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) BPI-Cys(S-mPEG)

BPI, which has a thiol-containing cysteine residue, is dissolved inbuffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-BPI Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock BPI solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The N^(ter) conjugate isisolated and according the general procedure outlined above.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) BPI-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock BPI solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The Lys conjugate is isolatedand purified according the general procedure outlined above to yield theBPI-Lys-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 16 C-peptide-mPEG Conjugates

C-peptide is a product of the cleavage of proinsulin, consisting of theB and A chains of insulin linked together via a connecting C-peptide,produced when proinsulin is released into the blood stream in responseto a rise in serum glucose. C-peptide has the sequence,Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln(U.S. Pat. No. 6,610,649). C-peptide alone has been proposed for thetreatment of diabetes (EP 132 769); insulin in combination withC-peptide can be administered for the prevention of diabeticcomplications (SE 460334).

a) mPEG-N^(ter)-C-peptide—Via mPEG-SPC

C-peptide is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of C-peptide, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of C-peptide prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-C-peptide conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) C-peptide-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of C-peptide, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedC-peptide (Prot-C-peptide, e.g.,Fmoc-Glu(tBu)-Ala-Glu(tBu)-Asp(tBu)-Leu-Gln-Val-Gly-Gln-Val-Glu(tBu)-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser(tBu)-Leu-Gln-Pro-Leu-Ala-Leu-Glu(tBu)-Gly-Ser(tBu)-Leu-Gln)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-C-peptide is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-C-peptide-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the C-peptide-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-C-peptide Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock C-peptide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) C-peptide-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of C-peptide, to provide a Glu-conjugateform of the peptide. For coupling to the Glu residue, a protectedC-peptide (Prot2 C-peptide, e.g.,Fmoc-Glu(tBu)-Ala-Glu(tBu)-Asp(tBu)-Leu-Gln-Val-Gly-Gln-Val-Glu(OBz)-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser(tBu)-Leu-Gln-Pro-Leu-Ala-Leu-Glu(tBu)-Gly-Ser(tBu)-Leu-Gln)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Glu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate forsubsequent coupling (Prot3-C-peptide, e.g.,Fmoc-Glu(tBu)-Ala-Glu(tBu)-Asp(tBu)-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser(tBu)-Leu-Gln-Pro-Leu-Ala-Leu-Glu(tBu)-Gly-Ser(tBu)-Leu-Gln)-O(tBu))mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-C-peptide isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-C-peptide-(Glu-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the C-peptide-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 17 Prosaptide™ TX14(A)-mPEG Conjugates

Prosaptide TX14(A) is a 14-mer amino acid sequence derived from theactive neurotrophic region in the amino-terminal portion of the saposinC domain. Prosaptides are active on a variety of neuronal cells,stimulating sulfatide synthesis and increasing sulfatide concentrationin Schwann cells and oligodendrocytes. This indicates that prosaposinand prosaptides are trophic factors for myelin formation. ProsaptideTX14(A) may have potential for therapeutic use in neuropathic painsyndromes in humans (Otero et al. Neurosci. Lett. 1999, 270, 29).Prosaptide TX14(A) is commercially available from AnaSpec (San Jose,Calif.) with the sequence,Thr-(D-Ala)-Leu-Ile-Asp-Asn-Asn-Ala-Thr-Glu-Glu-Ile-Leu-Tyr.

a) mPEG-N^(ter)-Prosaptide TX 14(A)—Via mPEG-SPC

An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of Prosaptide TX14(A), to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Prosaptide TX14(A) prepared in phosphate buffered saline,PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1M HCl, if necessary, to bring the pHof the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Prosaptide TX14(A) conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Prosaptide TX14(A)-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Prosaptide TX14(A), to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Prosaptide TX14(A) (Prot-Prosaptide TX14(A), e.g.,Fmoc-Thr(tBu)-(D-Ala)-Leu-Ile-Asp(tBu)-Asn-Asn-Ala-Thr(tBu)-Glu(tBu)-Glu(tBu)-Ile-Leu-Tyr(tBu)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Prosaptide TX14(A) is prepared in N,N-dimethylformamide is addedand the mixture is stirred using a magnetic stirrer until the mPEG-NH₂is fully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The conjugatesolution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine theextent of Prot-Prosaptide TX14(A)-C^(ter)-mPEG conjugate formation. Theremaining protecting groups are removed under standard deprotectionconditions to yield the Prosaptide TX14(A)-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Prosaptide TX14(A) Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Prosaptide TX14(A) solution and mixed well. After the addition ofthe mPEG-SMB, the pH of the reaction mixture is determined and adjustedto 6.7 to 6.8 using conventional techniques. To allow for coupling ofthe mPEG-SMB to the peptide via an amide linkage, the reaction solutionis stirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Prosaptide TX14(A)-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of Prosaptide TX14(A), to provide aGlu-conjugate form of the peptide. For coupling to the Glu residue, aprotected Prosaptide TX14(A) (Prot2-Prosaptide TX14(A), e.g.,Fmoc-Thr(tBu)-(D-Ala)-Leu-Ile-Asp(tBu)-Asn-Asn-Ala-Thr(tBu)-Glu(OBz)-Glu(tBu)-Ile-Leu-Tyr(tBu)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Glu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate forsubsequent coupling (Prot3-Prosaptide TX14(A), e.g.,Fmoc-Thr(tBu)-(D-Ala)-Leu-Ile-Asp(tBu)-Asn-Asn-Ala-Thr(tBu)-Glu-Glu(tBu)-Ile-Leu-Tyr(tBu)-O(tBu))mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-ProsaptideTX14(A) is prepared in N,N-dimethylformamide is added and the mixture isstirred using a magnetic stirrer until the mPEG-NH₂ is fully dissolved.The stirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Prosaptide TX14(A-(Glu-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Prosaptide TX14(A)-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 18 Sermorelin Acetate (GHRFA Group)-mPEG Conjugates

Sermorelin is the biologically active fragment of human growthhormone-releasing factor, consisting of GHRH (1-29)-amide, which can beused as a provocative test of growth hormone deficiency (Prakash andGoa, Biodrugs 1999, 12, 139). Sermoline may also increase IGF-1 levelsand improve body composition (increased lean mass and reduced truncaland visceral fat) in patients with HIV (Koutkia et al, JAMA 2004, 292,210). Synthetic sermorelin acetate is commercially available from GelacsInnovation (Hangzhou, China) with the sequence,Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH₂

a) mPEG-N^(ter)-Sermorelin—Via mPEG-SPC

An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of Sermorelin, to provide a N^(ter)-conjugateform of the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof Sermorelin prepared in phosphate buffered saline, PBS, pH 7.4 isadded and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Sermorelin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Sermorelin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Sermorelin, to provide a C^(a)-conjugateform of the peptide. For coupling to the C-terminus, a protectedSermorelin lacking the C-terminus amide (Prot-Sermorelin, e.g.,Fmoc-Tyr-Ala-Asp(tBu)-Ala-Ile-Phe-Thr-Asn-Ser(tBu)-Tyr(tBu)-Arg(Tos)-Lys-Val-Leu-Gly-Gln-Leu-Ser(tBu)-Ala-Arg(Tos)-Lys(Fmoc)-Leu-Leu-Gln-Asp(tBu)-Ile-Met-Ser(tBu)-Arg(Tos)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Sermorelin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Sermorelin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Sermorelin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Sermorelin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Sermorelin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Sermorelin-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Sermorelin (e.g.,Fmoc-Tyr-Ala-Asp(tBu)-Ala-Ile-Phe-Thr-Asn-Ser(tBu)-Tyr(tBu)-Arg(Tos)-Lys-Val-Leu-Gly-Gln-Leu-Ser(tBu)-Ala-Arg(Tos)-Lys-Leu-Leu-Gln-Asp(tBu)-Ile-Met-Ser(tBu)-Arg(Tos)-NH₂)solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theSermorelin-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 19 Pralmorelin-mPEG Conjugates

Pralmorelin (GHRP-2) is a growth-hormone releasing peptide having thecomposition,D-Ala-([3-(naphthalen-2-yl)]-D-Ala)-Ala-Trp-(D-Phe)-Lys-NH₂. Pralmorelinhas been proposed for the diagnosis of serious growth hormone deficiencyand for treatment of short stature (Furata et al. Arz.-Forsch. 2004, 54,868), and fro treating acute heart failure, chronic heart failure at aphase of acute exacerbation, and heart failure at a phase of transitionto chronic heart failure (U.S. Pat. No. 6,878,689).

a) mPEG-N^(ter)-Pralmorelin—Via mPEG-SPC

Pralmorelin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Pralmorelin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Pralmorelin prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Pralmoreling conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Pralmorelin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Pralmorelin, to provide a C^(a)-conjugateform of the peptide. For coupling to the C-terminus, a protectedPralmorelin lacking the C-terminus amide (Prot-Pralmorelin, e.g.,Fmoc-D-Ala-([3-(naphthalen-2-yl)]-D-Ala)-Ala-Trp-(D-Phe)-Lys(Fmoc)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Pralmorelin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Pralmorelin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Pralmorelin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-PP-Pralmorelin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Pralmorelin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Pralmorelin-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Pralmorelin (e.g.,Fmoc-D-Ala-([3-(naphthalen-2-yl)]-D-Ala)-Ala-Trp-(D-Phe)-Lys-NH₂)solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield thePralmorelin-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 20 Growth Hormone Releasing Factor (GHRFA Group)-mPEG Conjugates

Growth hormone-releasing factor (GHRF) is a hypothalamic peptide whichpositively regulates the synthesis and secretion of growth hormone inthe anterior pituitary. Growth hormone releasing factor is commerciallyavailable from GenScript Corporation (Piscataway, N.J.; Cat. No.RP10734) with the sequence,Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂(YADAIFTNSY RKVLGQLSAR KLLQDIMSRQ QGESNQERGA RARL-NH₂)

a) mPEG-N^(ter)-GHRF—Via mPEG-SPC

An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of GHRF, to provide a N^(ter)-conjugate formof the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof GHRF prepared in phosphate buffered saline, PBS, pH 7.4 is added andthe mixture is stirred using a magnetic stirrer until the mPEG-SPC isfully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The reaction isoptionally quenched to terminate the reaction. The pH of the conjugatesolution at the end of the reaction is measured and further acidified byaddition of 0.1 M HCl, if necessary, to bring the pH of the finalsolution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-GHRFconjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) GHRF—C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of GHRF, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected GHRF lackingthe C-terminus amide (Prot-GHRF, e.g.,Fmoc-Tyr(tBu)-Ala-Asp(tBu)-Ala-Ile-Phe-Thr(tBu)-Asn-Ser(tBu)-Tyr(tBu)-Arg(Tos)-Lys(Fmoc)-Val-Leu-Gly-Gln-Leu-Ser(tBu)-Ala-Arg(Tos)-Lys(Fmoc)-Leu-Leu-Gln-Asp(tBu)-Ile-Met-Ser(tBu)-Arg(Tos)-Gln-Gln-Gly-Glu(tBu)-Ser(tBu)-Asn-Gln-Glu(tBu)-Arg(Tos)-Gly-Ala-Arg(Tos)-Ala-Arg(Tos)-Leu-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-GHRF is prepared in N,N-dimethylformamide is added and the mixtureis stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-GHRF-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theGHRF-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-GHRF Via mPEG-SMB

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock GHRF solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) GHRF-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected GHRF (e.g.,Fmoc-Tyr(tBu)-Ala-Asp(tBu)-Ala-Ile-Phe-Thr(tBu)-Asn-Ser(tBu)-Tyr(tBu)-Arg(Tos)-Lys(Fmoc)-Val-Leu-Gly-Gln-Leu-Ser(tBu)-Ala-Arg(Tos)-Lys-Leu-Leu-Gln-Asp(tBu)-Ile-Met-Ser(tBu)-Arg(Tos)-Gln-Gln-Gly-Glu(tBu)-Ser(tBu)-Asn-Gln-Glu(tBu)-Arg(Tos)-Gly-Ala-Arg(Tos)-Ala-Arg(Tos)-Leu-NH₂)solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theGHRF-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 21 Examorelin (GHRFA Group)-mPEG Conjugates

Examorelin is a synthetic growth hormone releasing peptide; which hasbeen found to reverse the worsening of cardiac dysfunction in growthhormone deficient rats (Colonna et al., Eur. J. Pharmacol. 1997, 334,201), and has been suggested for the normalization of cardiac pressureand treating heart disease in humans (U.S. Pat. No. 5,932,548). Thesequence of Examorelin is His-(D-2-methyl-Trp)-Ala-Trp-(D-Phe)-Lys-NH₂.

a) mPEG-N^(ter)-Examorelin—Via mPEG-SPC

Examorelin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Examorelin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Examorelin prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Examorelin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Examorelin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Examorelin, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedExamorelin lacking the C-terminus amide (Prot-Examorelin, e.g.,Fmoc-His-(D-2-methyl-Trp)-Ala-Trp-(D-Phe)-Lys(Fmoc)-OH) is prepared andpurified according to standard automated peptide synthesis techniquesknown to those skilled in the art. mPEG-NH₂ 20 kDa, stored at −20° C.under argon, is warmed to ambient temperature. The reaction is performedat room temperature. About 3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-Examorelin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SD S-PAGE and RP-HPLC (C18) to determine the extent ofProt-Examorelin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Examorelin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Examorelin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Examorelin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Examorelin-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Examorelin (e.g.,Fmoc-His-(D-2-methyl-Trp)-Ala-Trp-(D-Phe)-Lys-NH₂) solution and mixedwell. After the addition of the mPEG-SMB, the pH of the reaction mixtureis determined and adjusted to 6.7 to 6.8 using conventional techniques.To allow for coupling of the mPEG-SMB to the peptide via an amidelinkage, the reaction solution is stirred for several hours (e.g., 5hours) at room temperature in the dark or stirred overnight at 3-8° C.in a cold room, thereby resulting in a conjugate solution. The reactionis quenched with a 20-fold molar excess (with respect to the peptide) ofTris buffer. The remaining protecting groups are removed under standarddeprotection conditions to yield the Examorelin-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 22 Gonadorelin (LH-Related Peptide Group)-mPEG Conjugates

Gonadorelin (GnRH) is a decapeptide that stimulates the synthesis andsecretion of both pituitary gonadotropins, luteinizing hormone andfollicle stimulating hormone. GnRH is produced by neurons in the septumpreoptic area of the hypothalamus and released into the pituitary portalblood, leading to stimulation of gonadotrophs in the anterior pituitarygland. Gonadorelin has been proposed for treating benign prostatichyperplasia (U.S. Pat. No. 4,321,260), prostatic hypertrophy (U.S. Pat.No. 5,610,136), treating malignant neoplasia and acquired immunedeficiency syndrome (U.S. Pat. No. 4,966,753), management of prostateand breast carcinoma, endometriosis and uterine leiomyomata, precociouspuberty and nontumorous ovarian hyperandrogenic syndromes (Pace et al.,Am. Fam. Physician 1991, 44, 1777). Synthetic gonadorelin iscommercially available from Gelacs Innovation (Hangzhou, China) with thesequence, Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂.

a) GnRH-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of GnRH, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected GnRH lackingthe C-terminus amide (Prot-GnRH, e.g.,Glp-His-Trp-Ser(tBu)-Tyr(tBu)-Gly-Leu-Arg(Tos)-Pro-Gly-OH) is preparedand purified according to standard automated peptide synthesis orrecombinant techniques known to those skilled in the art. mPEG-NH₂ 20kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. About 3-5-fold molarexcess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-GnRH is preparedin N,N-dimethylformamide is added and the mixture is stirred using amagnetic stirrer until the mPEG-NH₂ is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The conjugate solution is then analyzed by SDS-PAGEand RP-HPLC (C18) to determine the extent of Prot-GnRH-C^(ter)-mPEGconjugate formation. The remaining protecting groups are removed understandard deprotection conditions to yield the GnRH-C^(ter)-mPEGconjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 23 Corticoliberin-mPEG conjugates

Corticoliberin is a 41-amino acid peptide hormone and neutrotransmitterinvolved in the stress response having the sequence,Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala(Vale et al., Science 1981, 4514, 1394). In humans, CRH regulates, viarelease of proopiomelanocortin, ACTH secretion from the anteriorpituitary and has several direct actions on central and peripheraltissues. Corticoliberin has also been found to have directanti-inflammatory properties. Thus, corticoliberin has found therapeuticuses inhibiting inflammatory response (U.S. Pat. No. 4,801,612), andreduction of edema for brain and musculature injury (U.S. Pat. No.5,137,871), i.e., the use of CRH to decrease the leakage of bloodcomponents into tissues produced by various adverse medical conditions,and thus to treat a patient for injury to or disease of the brain,central nervous system or musculature in which edema is a factor.

a) mPEG-N^(ter) Corticoliberin—Via mPEG-SPC

Corticoliberin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Corticoliberin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Corticoliberin prepared in phosphate buffered saline, PBS,pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1 M HCl, if necessary, to bring thepH of the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Corticoliberin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Corticoliberin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Corticoliberin, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Corticoliberin (Prot-Corticoliberin, e.g.,Fmoc-Ser(tBu)-Gln-Glu(tBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(tBu)-Leu-Thr(tBu)-Phe-His-Leu-Leu-Arg(Tos)-Glu(tBu)-Val-Leu-Glu(tBu)-Met-Thr(tBu)-Lys(Fmoc)-Ala-Asp(tBu)-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser(tBu)-Asn-Arg(Tos)-Lys(Fmoc)-Leu-Leu-Asp(tBu)-Ile-Ala)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Corticoliberin is prepared in N,N-dimethylformamide is added andthe mixture is stirred using a magnetic stirrer until the mPEG-NH₂ isfully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The conjugatesolution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine theextent of Prot-Corticoliberin-C^(ter)-mPEG conjugate formation. Theremaining protecting groups are removed under standard deprotectionconditions to yield the Corticoliberin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Corticoliberin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Corticoliberin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Corticoliberin-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Corticoliberin (e.g.,Fmoc-Ser(tBu)-Gln-Glu(tBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(tBu)-Leu-Thr(tBu)-Phe-His-Leu-Leu-Arg(Tos)-Glu(tBu)-Val-Leu-Glu(tBu)-Met-Thr(tBu)-Lys-Ala-Asp(tBu)-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser(tBu)-Asn-Arg(Tos)-Lys(Fmoc)-Leu-Leu-Asp(tBu)-Ile-Ala-O(tBu))solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theCorticoliberin-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 24 Atrial Natriuretic Peptide (Atriopeptin)-mPEG Conjugates

Atrial natriuretic peptide (ANP; atriopeptin) is a peptide hormonesecreted by muscle cells in the upper atria of the heart, in response tohigh blood pressure. It is involved in the homeostatic control of bodywater, sodium, potassium and adiposity. ANP acts to reduce the water,sodium and adipose loads on the circulatory system, thereby reducingblood pressure (Needleman and Greenwald, N. Engl. J. Med. 1986, 314,828). Human atrial natriuretic peptide is commercially available fromGenScript Corporation (Piscataway, N.J.; Cat. No. RP 11927) with thesequence,Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr(SLRRSSCFGG RMDRIGAQSG LGCNSFRY).

a) mPEG-N^(ter)-ANP—Via mPEG-SPC

An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of ANP, to provide a N^(ter)-conjugate formof the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof ANP prepared in phosphate buffered saline, PBS, pH 7.4 is added andthe mixture is stirred using a magnetic stirrer until the mPEG-SPC isfully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The reaction isoptionally quenched to terminate the reaction. The pH of the conjugatesolution at the end of the reaction is measured and further acidified byaddition of 0.1M HCl, if necessary, to bring the pH of the finalsolution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-ANPconjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) ANP-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of ANP, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected ANP(Prot-ANP, e.g.,Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(tBu)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-ANP is prepared in N,N-dimethylformamide is added and the mixtureis stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-ANP-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theANP-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) ANP-Cys(S-mPEG)

ANP, which has a thiol-containing cysteine residue, is dissolved inbuffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-ANP Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock ANP solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) ANP-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of ANP, to provide a Asp-conjugate form ofthe peptide. For coupling to the Asp residue, a protected ANP(Prot2-ANP, e.g.,Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(OBz)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-ANP, e.g.,Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-O(tBu)).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-ANP is preparedin N,N-dimethylformamide is added and the mixture is stirred using amagnetic stirrer until the mPEG-NH₂ is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The conjugate solution is then analyzed by SDS-PAGEand RP-HPLC (C18) to determine the extent of Prot3-ANP-(Asp-O-mPEG)conjugate formation. The remaining protecting groups are removed understandard deprotection conditions to yield the ANP-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 25 AnergiX-mPEG Conjugates

AnergiX is a T cell inhibitor which has been proposed for the treatmentof rheumatoid arthritis comprising soluble Major HistocompatibilityComplex (MHC) molecules linked to antigenic peptides recognized byspecific subsets of T cells (U.S. Pat. No. 5,468,481).

a) mPEG-N^(ter)-AnergiX Via mPEG-SPC

AnergiX is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of AnergiX, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of AnergiX prepared in phosphate buffered saline, PBS, pH 7.4is added and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-AnergiX conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) AnergiX-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of AnergiX, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protected AnergiX(Prot-AnergiX) is prepared and purified according to standard automatedpeptide synthesis techniques known to those skilled in the art. mPEG-NH₂20 kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. About 3-5-fold molarexcess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-AnergiX isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SD S-PAGE and RP-HPLC (C18) to determine the extent ofProt-AnergiX-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theAnergiX-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-AnergiX Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock AnergiX solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 26 Somatostatin-mPEG Conjugates

Somatostatin is a peptide hormone that regulates the endocrine systemand affects neurotransmission and cell proliferation via interactionwith G-protein-coupled somatostatin receptors (of which five differentsubtypes have been characterized) and inhibition of the release ofnumerous secondary hormones. Binding to the different types ofsomatostatin subtypes have been associated with the treatment of variousconditions and/or diseases. (Raynor et al., Molecular Pharmacol. 1993,43, 838; Lloyd, et al., Am. J. Physiol. 1995, 268, G102) Indicationsassociated with activation of the somatostatin receptor subtypes areinhibition of insulin and/or glucagon for treating diabetes mellitus,angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy;inhibition of gastric acid secretion and more particularly pepticulcers, enterocutaneous and pancreaticocutaneous fistula, irritablebowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS relateddiarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitisand gastrointestinal hormone secreting tumors; treatment of cancer suchas hepatoma; inhibition of angiogenesis, treatment of inflammatorydisorders such as arthritis; retinopathy; chronic allograft rejection;angioplasty; preventing graft vessel and gastrointestinal bleeding.Somatostatin is commercially available from Gelacs Innovation (Hangzhou,China)) with the sequence,His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH₂

a) mPEG-N^(ter)—Somatostatin—Via mPEG-SPC

An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of Somatostatin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Somatostatin prepared in phosphate buffered saline, PBS,pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1M HCl, if necessary, to bring the pHof the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Somatostatin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Somatostatin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Somatostatin, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Somatostatin lacking the C-terminus amide(Prot-Somatostatin,e.g.,Fmoc-His-Ser(tBu)-Asp(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(tBu)-Leu-Ser(tBu)-Arg(Tos)-Leu-Arg(Tos)-Asp(tBu)-Ser(tBu)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Somatostatin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Somatostatin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Somatostatin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)—Somatostatin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Somatostatin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Somatostatin-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of Somatostatin, to provide a Asp-conjugateform of the peptide. For coupling to the Asp residue, a protectedSomatostatin (Prot2-Somatostatin, e.g.,Fmoc-His-Ser(tBu)-Asp(OBz)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(tBu)-Leu-Ser(tBu)-Arg(Tos)-Leu-Arg(Tos)-Asp(tBu)-Ser(tBu)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-NH₂)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-Somatostatin, e.g.,Fmoc-His-Ser(tBu)-Asp-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(tBu)-Leu-Ser(tBu)-Arg(Tos)-Leu-Arg(Tos)-Asp(tBu)-Ser(tBu)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-_(NH2)).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Somatostatin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Somatostatin-(Asp-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Somatostatin-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 27 29-Amino-Acid Peptide Growth Hormone Releasing Hormone (GHRH)Analogue-mPEG Conjugates

GHRH stimulates growth hormone secretion from the anterior pituitarygland. GHRH can increase the height velocity in children with growthdisorders (idiopathic growth hormone deficiency). In addition, GHRH canhelp production of muscle mass and stimulate fat breakdown bystimulating indirectly the production of IGF-1 via inducing the releaseof growth hormone. Most patients with idiopathic growth hormonedeficiency have a deficit in hypothalamic GHRH synthesis or releaserather than in growth hormone itself, so treatment with GHRH isconsidered a logical approach in the management of these patients. GHRHhas a very short half-life (10-20 min) due to rapid proteolysis andglomerular filtration. The 29-amino acid peptide of GHRH (“GHRH-29”) hasthe sequenceTyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Glu-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg.

a) mPEG-N^(ter)-GHRH-29 Via mPEG-SPC

GHRH-29 is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of GHRH-29, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of GHRH-29 prepared in phosphate buffered saline, PBS, pH 7.4is added and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-GHRH-29 conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) GHRH-29-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of GHRH-29, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protected GHRH-29(Prot-GHRH-29) is prepared and purified according to standard automatedpeptide synthesis techniques known to those skilled in the art. mPEG-NH₂20 kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. About 3-5-fold molarexcess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-GHRH-29 isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-GHRH-29-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theGHRH-29-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-GHRH-29 Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock GHRH-29 solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 28 Bremelanotide (Melanocortin Agonist Group)-mPEG Conjugates

Bremelanotide is a cyclic hepta-peptide lactam analog ofalpha-melanocyte-stimulating hormone (alpha-MSH) that activates themelanocortin receptors MC3-R and MC4-R in the central nervous system. Ithas been proposed for use in treating sexual dysfunction in men(erectile dysfunction or impotence) as well as sexual dysfunction inwomen (sexual arousal disorder). Bremelanotide has the sequence(NAc-Nle)-cyclo[Asp-His-(D-Phe)-Arg-Trp-Lys]-OH (U.S. Pat. Nos.6,579,968 and 6,794,489).

a) mPEG-N^(ter)-Bremelanotide—Via mPEG-SPC

Bremelanotide lacking the N-terminus acetyl group (NH₂-Bremelanotide) isprepared and purified according to standard automated peptide synthesisor recombinant techniques known to those skilled in the art. Anillustrative polymeric reagent, mPEG-SPC reagent, is covalently attachedto the N-terminus of Bremelanotide, to provide a N^(ter)-conjugate formof the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof NH₂-Bremelanotide prepared in phosphate buffered saline, PBS, pH 7.4is added and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Bremelanotide conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Bremelanotide-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Bremelanotide, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Bremelanotide (Prot-Bremelanotide, e.g., sequence(NAc-Nle)-cyck[Asp-His-(D-Phe)-Arg(Tos)-Trp-Lys]-OH) is prepared andpurified according to standard automated peptide synthesis techniquesknown to those skilled in the art. mPEG-NH₂ 20 kDa, stored at −20° C.under argon, is warmed to ambient temperature. The reaction is performedat room temperature. About 3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-Bremelanotide isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Bremelanotide-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Bremelanotide-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Bremelanotide Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Bremelanotide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 29 Melanocortin Peptidomimetic Compound (Melanocortin AgonistGroup)-mPEG Conjugates

Melanocortins are a group of pituitary peptide hormones that includeadrenocortinotropin (ACTH) and the alpha, beta and gammamelanocyte-stimulating hormones (MSH) that derive from the prohormoneproopiomelanocortin. Melanocortins act through several melanocortinreceptors designated MC-1 through MC5. Several synthetic melanocortinsare in development including Palatin Technologies' bremelanotide forerectile dysfunction and sexual arousal disorder. Bremelanotide is acyclic hepta-peptide lactam analog of alpha-melanocyte-stimulatinghormone that activates MC-3 and MC-4. Bremelanotide acts within thecentral nervous system rather than the vascular system (blood flow) toelicit arousal. The peptide has the amino acid sequenceAc-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH.

a) mPEG-N^(ter)-Bremelanotide Via mPEG-SPC

Bremelanotide is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of Bremelanotide, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Bremelanotide prepared in phosphate buffered saline, PBS,pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1M HCl, if necessary, to bring the pHof the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Bremelanotide conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Bremelanotide-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Bremelanotide, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Bremelanotide (Prot-Bremelanotide) is prepared and purifiedaccording to standard automated peptide synthesis techniques known tothose skilled in the art. mPEG-NH₂ 20 kDa, stored at −20° C. underargon, is warmed to ambient temperature. The reaction is performed atroom temperature. About 3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-Bremelanotide isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Bremelanotide-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Bremelanotide-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Bremelanotide Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Bremelanotide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 30 Recombinant LH (Luteinizing Hormone) (LH-Related PeptideGroup)-mPEG Conjugates

LH appears to play an important role in both male and femalereproduction. In females, an LH surge is associated with ovulation andwith the initiation of the conversion of the residual follicle into acorpus luteum that, in turn, produces progesterone to prepare theendometrium for a possible implantation. In males, through the Leydigcell of the testes, LH is responsible for the production oftestosterone. LH is a glycoprotein composed of two subunits attached viatwo disulfide bonds. The two subunits are comprised of 92 and 121 aminoacids.

a) mPEG-N^(ter)-LH Via mPEG-SPC

LH is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of LH, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of LH prepared in phosphate buffered saline, PBS, pH 7.4 isadded and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-LHconjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) LH-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of LH, to provide a C^(ter)-conjugate form ofthe peptide. For coupling to the C-terminus, a protected LH (Prot-LH) isprepared and purified according to standard automated peptide synthesistechniques known to those skilled in the art. mPEG-NH₂ 20 kDa, stored at−20° C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-NH₂,PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-LH is prepared in N,N-dimethylformamide is added and the mixture isstirred using a magnetic stirrer until the mPEG-NH₂ is fully dissolved.The stirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-LH-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theLH-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-LH Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock LH solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 31 Terlipressin-mPEG Conjugates

Terlipressin is an analogue of vasopressin used as a vasoactive drug inthe management of hypotension. It has been found to be effective whennorepinephrine does not help. Indications for use includenorepinephrine-resistant septic shock (O'Brien et al., Lancet, 2002,359, 1209), hepatorenal syndrome (Gluud et al., Cochrane Database ofSystematic Reviews 2006, Issue 3. Art. No.: CD005162. DOI:10.1002/14651858.CD005162.pub2) and bleeding esophageal varices (Ioannouet al., Cochrane Database of Systematic Reviews 2003, Issue 1. Art. No.:CD002147. DOI: 10.1002/14651858.CD002147). Terlipressin has thesequence, Gly-Gly-Gly-cyclo-[Cys-Tyr-Phe-Gln-Asp-Cys]-Pro-Lys-GlyNH₂.

a) mPEG-N^(ter)-Terlipressin—Via mPEG-SPC

Terlipressin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Terlipressin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Terlipressin prepared in phosphate buffered saline, PBS,pH 7.4 is added and the mixture is stirred using a magnetic stirreruntil the mPEG-SPC is fully dissolved. The stirring speed is reduced andthe reaction is allowed to proceed to formation of conjugate product.The reaction is optionally quenched to terminate the reaction. The pH ofthe conjugate solution at the end of the reaction is measured andfurther acidified by addition of 0.1 M HCl, if necessary, to bring thepH of the final solution to about 5.5. The conjugate solution is thenanalyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Terlipressin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Terlipressin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Terlipressin, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Terlipressin lacking the C-terminus amide (Prot-Terlipressin,e.g.,Fmoc-Gly-Gly-Gly-Cys(tBu)-Tyr(tBu)-Phe-Gln-Asp(tBu)-Cys(tBu)-Pro-Lys(Fmoc)-Gly-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Terlipressin is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Terlipressin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Terlipressin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Terlipressin-Cys(S-mPEG)

Terlipressin, which has a thiol-containing cysteine residue, isdissolved in buffer. To this peptide solution is added a 3-5 fold molarexcess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperatureunder an inert atmosphere for several hours. Analysis of the reactionmixture reveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Terlipressin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Terlipressin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Terlipressin-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Terlipressin (e.g.,Fmoc-Gly-Gly-Gly-Cys(tBu)-Tyr(tBu)-Phe-Gln-Asp(tBu)-Cys(tBu)-Pro-Lys-Gly-NH₂)solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theTerlipressin-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 32 Ecallantide-mPEG Conjugates

Ecallantide is a 60-amino acid peptide which is an inhibitor of theprotein kallikrein used for hereditary angioedema and in the preventionof blood loss in cardiothoracic surgery (Lehmann, Expert Opin. Biol.Ther. 2008, 8, 1187). It has been shown to inhibit kallikrein in alaboratory investigation known as phage display (Lehmann, 2008).Ecallantide has the sequenceGlu-Ala-Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Asp-Asp-Gly-Pro-Cys-Arg-Ala-Ala-His-Pro-Arg-Trp-Phe-Phe-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Glu-Glu-Phe-Ile-Tyr-Gly-Gly-Cys-Glu-Gly-Asn-Gln-Asn-Arg-Phe-Glu-Ser-Leu-Glu-Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp(U.S. Pat. Appl. Pub. No. 20070213275).

a) mPEG-N^(ter)-Ecallantide—Via mPEG-SPC

Ecallantide is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Ecallantide, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Ecallantide prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Ecallantide conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Ecallantide-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Ecallantide, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Ecallantide (Prot-Ecallantide, e.g.,Fmoc-Glu(tBu)-Ala-Met-His-Ser(tBu)-Phe-Cys(tBu)-Ala-Phe-Lys(Fmoc)-Ala-Asp(tBu)-Asp(tBu)-Gly-Pro-Cys(tBu)-Arg(Tos)-Ala-Ala-His-Pro-Arg(Tos)-Trp-Phe-Phe-Asn-Ile-Phe-Thr(tBu)-Arg(Tos)-Gln-Cys(tBu)-Glu(tBu)-Glu(tBu)-Phe-Ile-Tyr(tBu)-Gly-Gly-Cys(tBu)-Glu(tBu)-Gly-Asn-Gln-Asn-Arg(Tos)-Phe-Glu(tBu)-Ser(tBu)-Leu-Glu(tBu)-Glu(tBu)-Cys(tBu)-Lys(Fmoc)-Lys(Fmoc)-Met-Cys(tBu)-Thr(tBu)-Arg(Tos)-Asp(tBu)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Ecallantide is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Ecallantide-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Ecallantide-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Ecallantide-Cys(S-mPEG)

Ecallantide, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-PP-Ecallantide Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Ecallantide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Ecallantide-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Ecallantide (e.g.,Fmoc-Glu(tBu)-Ala-Met-His-Ser(tBu)-Phe-Cys(tBu)-Ala-Phe-Lys-Ala-Asp(tBu)-Asp(tBu)-Gly-Pro-Cys(tBu)-Arg(Tos)-Ala-Ala-His-Pro-Arg(Tos)-Trp-Phe-Phe-Asn-Ile-Phe-Thr(tBu)-Arg(Tos)-Gln-Cys(tBu)-Glu(tBu)-Glu(tBu)-Phe-Ile-Tyr(tBu)-Gly-Gly-Cys(tBu)-Glu(tBu)-Gly-Asn-Gln-Asn-Arg(Tos)-Phe-Glu(tBu)-Ser(tBu)-Leu-Glu(tBu)-Glu(tBu)-Cys(tBu)-Lys(Fmoc)-Lys(Fmoc)-Met-Cys(tBu)-Thr(tBu)-Arg(Tos)-Asp(tBu)-NH₂)solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theEcallantide-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 33 Calphobindin I-mPEG Conjugates

Calphobindin I (CPB-I, annexin V) is an anticoagulant protein purifiedfrom human placenta; it is a member of the annexin family that bindsphospholipids in a calcium-dependent manner. CPB-I helpsreepithelialization through the promotion of both uPA synthesis andmigration of keratinocytes without stimulating their proliferation(Nakao et al., Eur. J. Biochem. 2005, 223, 901). Calphobindin I has thesequence, Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe AspGlu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly Thr AspGlu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln GluIle Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu LysSer Glu Leu Thr Gly Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro SerArg Leu Tyr Asp Ala Tyr Glu Leu Lys His Ala Leu Lys Gly Ala Gly Thr AsnGlu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg AlaIle Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val ValGly Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala AsnArg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln AlaLeu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile ThrIle Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr MetThr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile Asp Arg Glu Thr Ser Gly AsnLeu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Ala TyrLeu Ala Glu Thr Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His ThrLeu Ile Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg LysGlu Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp ThrSer Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp (U.S.Pat. No. 7,393,833).

a) mPEG-N^(ter)-CPB-I—Via mPEG-SPC

CPB-I is prepared and purified according to standard recombinanttechniques known to those skilled in the art. An illustrative polymericreagent, mPEG-SPC reagent, is covalently attached to the N-terminus ofCPB-I, to provide a N^(ter)-conjugate form of the peptide. mPEG-SPC 20kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. About 3-5-fold molarexcess of mPEG-SPC 20 kDa reagent is used based upon absolute peptidecontent. The mPEG-SPC reagent is weighed into a glass vial containing amagnetic stirrer bar. A solution of CPB-I prepared in phosphate bufferedsaline, PBS, pH 7.4 is added and the mixture is stirred using a magneticstirrer until the mPEG-SPC is fully dissolved. The stirring speed isreduced and the reaction is allowed to proceed to formation of conjugateproduct. The reaction is optionally quenched to terminate the reaction.The pH of the conjugate solution at the end of the reaction is measuredand further acidified by addition of 0.1M HCl, if necessary, to bringthe pH of the final solution to about 5.5. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-CPB-I conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) CPB-I-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of CPB-I, to provide a C^(ter)-conjugate formof the peptide. mPEG-NH₂ 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of CPB-I is prepared inN,N-dimethylformamide is added and the mixture is stirred using amagnetic stirrer until the mPEG-NH₂ is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The conjugate solution is then analyzed by SDS-PAGEand RP-HPLC (C18) to determine the extent of CPB-I-C^(ter)-mPEGconjugate formation. The C^(ter) conjugate is isolated and purifiedaccording the general procedure outlined above.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) CPB-I-Cys(S-mPEG)

CPB-I, which has a thiol-containing cysteine residue, is dissolved inbuffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-CPB-I Via mPEG-SMB

mPEG-SMB, 510a, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock CPB-I solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) CPB-I-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock CPB-I solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The Lys conjugate is isolatedand purified according the general procedure outlined above to yield theCPB-I-Lys-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 34 Tiplimotide-mPEG Conjugates

Tiplimotide is the myelin basic protein peptide, amino acid sequence83-99,D-Ala-Lys-Pro-Val-Val-His-Leu-Phe-Ala-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro,(U.S. Pat. No. 6,379,670). Subcutaneous administration of tiplimotide inmultiple sclerosis patients can induce an APL-reactive immune responsein which T lymphocytes cross-reactive with the immunodominantneuroantigen MBP secrete anti-inflammatory cytokines (Crowe et al., Ann.Neurol. 2000, 48, 758).

a) mPEG-N^(ter)-Tiplimotide—Via mPEG-SPC

Tiplimotide is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Tiplimotide, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Tiplimotide prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Tiplimotide conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Tiplimotide-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Tiplimotide, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Tiplimotide (Prot-Tiplimotide, e.g.,Fmoc-D-Ala-Lys(Fmoc)-Pro-Val-Val-His-Leu-Phe-Ala-Asn-Ile-Val-Thr(tBu)-Pro-Arg(Tos)-Thr(tBu)-Pro)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Tiplimotide is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Tiplimotide-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Tiplimotide-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Tiplimotide Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Tiplimotide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Tiplimotide-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Tiplimotide (e.g.,Fmoc-D-Ala-Lys-Pro-Val-Val-His-Leu-Phe-Ala-Asn-Ile-Val-Thr(tBu)-Pro-Arg(Tos)-Thr(tBu)-Pro-O(tBu))solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theTiplimotide-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 35 Osteogenic Growth Peptide-mPEG Conjugates

Osteogenic growth peptide (OGP) is a circulating stimulator ofosteoblastic activity; identical to the C-terminus of histone H4 havingthe sequence, Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-Gly(PubChem Compound ID: 16132186). In particular, osteogenic growthpeptide has been shown to have a regulatory role in bone formation andhemopoiesis (Bab and Chorev, Biopolymers 2002, 66, 33).

a) mPEG-N^(ter)-OGP—Via mPEG-SPC

OGP is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of OGP, to provide a N^(ter)-conjugate formof the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof OGP prepared in phosphate buffered saline, PBS, pH 7.4 is added andthe mixture is stirred using a magnetic stirrer until the mPEG-SPC isfully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The reaction isoptionally quenched to terminate the reaction. The pH of the conjugatesolution at the end of the reaction is measured and further acidified byaddition of 0.1M HCl, if necessary, to bring the pH of the finalsolution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-OGPconjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) OGP-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of OGP, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected OGP(Prot-OGP, e.g.,Fmoc-Ala-Leu-Lys(Fmoc)-Arg(Tos)-Gln-Gly-Arg(Boc)-Thr(tBu)-Leu-Tyr(tBu)-Gly-Phe-Gly-Gly)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-OGP is prepared in N,N-dimethylformamide is added and the mixtureis stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-OGP-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theOGP-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-OGP Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock OGP solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) OGP-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected OGP (e.g.,Fmoc-Ala-Leu-Lys-Arg(Tos)-Gln-Gly-Arg(Boc)-Thr(tBu)-Leu-Tyr(tBu)-Gly-Phe-Gly-Gly-O(tBu))solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theOGP-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 36 Myelin Basic Protein-mPEG Conjugates

Myelin basic protein (MBP) is believed to be important in the process ofmyelination of nerves in the central nervous system. Myelin basicprotein (MBP) binds to the cytosolic surface of oligodendrocytemembranes via negatively charged lipids and is responsible for adhesionof these surfaces in the multilayered myelin sheath (Musse and Harauz,Int. Rev. Neurobiol. 2007, 79, 149). Human myelin basic protein has thesequence, MASQKRPSQR HGSKYLATAS TMDHARHGFL PRHRDTGILD SIGRFFGGDRGAPKRGSGKV PWLKPGRSPL PSHARSQPGL CNMYKDSHHP ARTAHYGSLP QKSHGRTQDENPVVHFFKNI VTPRTPPPSQ GKGRGLSLSR FSWGAEGQRP GFGYGGRASD YKSAHKGFKGVDAQGTLSKI FKLGGRDSRS GSPMARR (PubChem Protein Accession No. CAA351749;Streicher and Stoffel, Biol. Chem. Hoppe-Seyler 1989, 370 (5), 503).

a) mPEG-N^(ter)-MBP—Via mPEG-SPC

MBP is prepared and purified according to standard recombinanttechniques known to those skilled in the art. An illustrative polymericreagent, mPEG-SPC reagent, is covalently attached to the N-terminus ofMBP, to provide a N^(ter)-conjugate form of the peptide. mPEG-SPC 20kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. About 3-5-fold molarexcess of mPEG-SPC 20 kDa reagent is used based upon absolute peptidecontent. The mPEG-SPC reagent is weighed into a glass vial containing amagnetic stirrer bar. A solution of MBP prepared in phosphate bufferedsaline, PBS, pH 7.4 is added and the mixture is stirred using a magneticstirrer until the mPEG-SPC is fully dissolved. The stirring speed isreduced and the reaction is allowed to proceed to formation of conjugateproduct. The reaction is optionally quenched to terminate the reaction.The pH of the conjugate solution at the end of the reaction is measuredand further acidified by addition of 0.1M HCl, if necessary, to bringthe pH of the final solution to about 5.5. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-MBP conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) MBP-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of MBP, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, mPEG-NH₂ 20 kDa, storedat −20° C. under argon, is warmed to ambient temperature. The reactionis performed at room temperature. About 3-5-fold molar excess ofmPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution of MBPis prepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofMBP-C^(ter)-mPEG conjugate formation. The C^(ter) conjugate is isolatedand purified according the general procedure outlined above.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) MBP-Cys(S-mPEG)

MBP, which has a thiol-containing cysteine residue, is dissolved inbuffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-MBP Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock MBP solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) MBP-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock MBP solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The Lys conjugate is isolatedand purified according the general procedure outlined above to yield theMBP-Lys-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 37 Dynorphin A-mPEG Conjugates

Dynorphin A is a member of a class of opiod peptides that arise fromcleavage of a precursor protein, prodynorphin and is a 17 amino acidpeptide having the sequence,Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln(PubChem Substance ID No. 4731). Dynorphins primarily exert theireffects through the κ-opioid receptor (KOR), a G-protein coupledreceptor and have been shown to play a role as central nervous systemtransmitters. Dynorphin A has been proposed for uses including thesuppression of the cytotoxic activity of mammalian Natural Killer (NK)cells in recipients of transplanted tissue and individuals sufferingfrom autoimmune diseases (U.S. Pat. No. 5,817,628).

a) mPEG-N^(ter)-Dynorphin A—Via mPEG-SPC

Dynorphin A is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Dynorphin A, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Dynorphin A prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Dynorphin A conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Dynorphin A-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Dynorphin A, to provide aC^(ter)-conjugate form of the peptide. For coupling to the C-terminus, aprotected Dynorphin A (Prot-Dynorphin A, e.g.,Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu-Arg(Tos)-Arg(Tos)-Ile-Arg(Tos)-Pro-Lys(Fmoc)-Leu-Lys(Fmoc)-Trp-Asp(tBu)-Asn-Gln)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Dynorphin A is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Dynorphin A-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Dynorphin A-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Dynorphin A Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Dynorphin A solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Dynorphin A-Lys-mPEG

PEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protected Dynorphin A (e.g.,Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu-Arg(Tos)-Arg(Tos)-Ile-Arg(Tos)-Pro-Lys-Leu-Lys(Fmoc)-Trp-Asp(tBu)-Asn-Gln-O(tBu))solution and mixed well. After the addition of the mPEG-SMB, the pH ofthe reaction mixture is determined and adjusted to 6.7 to 6.8 usingconventional techniques. To allow for coupling of the mPEG-SMB to thepeptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer. The remaining protecting groupsare removed under standard deprotection conditions to yield theDynorphin A-Lys(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

Example 38 Anaritide (Natriuretic Peptide Group)-mPEG Conjugates

Anaritide is an antihypertensive 25-amino-acid synthetic form of atrialnatriuretic peptide used in the treatment of oliguric acute renalfailure having the sequence,Arg-Ser-Ser-cyclo-(Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys)-Asn-Ser-Phe-Arg-Tyr.

a) mPEG-M″-Anaritide—Via mPEG-SPC

Anaritide is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Anaritide, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Anaritide prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Anaritide conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Anaritide-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Anaritide, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedAnaritide (Prot-Anaritide, e.g.,Fmoc-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(tBu)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-Anaritide is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Anaritide-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Anaritide-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Anaritide-Cys(S-mPEG)

Anaritide, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Anaritide Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Anaritide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Anaritide-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of Anaritide, to provide an Asp-conjugateform of the peptide. For coupling to the Asp residue, a protectedAnaritide (Prot2-Anaritide, e.g.,Fmoc-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(OBz)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-Anaritide, e.g.,Fmoc-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-O(tBu)).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Anaritide isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Anaritide-(Asp-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Anaritide-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety

Example 39 Secretin-mPEG Conjugates

Secretin a 27 amino acid peptide hormone produced in the S cells of theduodenum in the crypts of Lieberkühn to primarily regulate the pH of theduodenal contents via the control of gastric acid secretion andbuffering with bicarbonate. Secretin is commercially available fromGelacs Innovation (Hangzhou, China) with the sequence,His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH₂.

a) mPEG-N^(ter)-Secretin—Via mPEG-SPC

Secretin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent, iscovalently attached to the N-terminus of Secretin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Secretin prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Secretin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Secretin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Secretin, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedSecretin (Prot-Secretin, e.g.,Fmoc-His-Ser(tBu)-Asp(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(tBu)-Leu-Ser(tBu)-Arg(Tos)-Leu-Arg(Tos)-Asp(tBu)-Ser(tBu)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-OH)is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. About3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-Secretin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Secretin-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theSecretin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Secretin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Secretin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Secretin-Asp(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Asp residue of Secretin to provide a Asp-conjugate formof the peptide. For coupling to the Asp residue, a protected Secretin(Prot2-Secretin, e.g.,Fmoc-His-Ser(tBu)-Asp(OBz)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(tBu)-Leu-Ser(tBu)-Arg(Tos)-Leu-Arg(Tos)-Asp(tBu)-Ser(tBu)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-NH₂)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Asp(OBz) residue (H₂/Pd) yields the free-Asp carboxylate forsubsequent coupling (Prot3-Secretin, e.g.,Fmoc-His-Ser(tBu)-Asp-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(tBu)-Leu-Ser(tBu)-Arg(Tos)-Leu-Arg(Tos)-Asp(tBu)-Ser(tBu)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-NH₂).mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Secretin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Secretin-(Asp-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Secretin-Asp(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety

Example 40 GLP-2-mPEG Conjugates

GLP-2 is a 33 amino acid peptide with the sequenceHis-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Tyr-Asp(HADGSFSDEM NTILDNLAAR DFINWLIQTK ITDR, available from GenScriptCorporation, Piscataway, N.J.; Cat. No. RP10774) which is produced bythe post-translational cleavage or proglucagon in the intestinalendocrine L cells and neurons of the central nervous system. GLP-2 hasbeen proposed for treatments for short bowel syndrome (Jeppesen et al.,Gastroenterology 2001, 120, 806), Crohn's disease (Peyrin-Biroulet etal., Lancet 2008, 372, 67) and osteroporosis (U.S. Pat. No. 6,943,151).

a) mPEG-N^(ter)-GLP-2—Via mPEG-SPC

GLP-2 is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent, is covalentlyattached to the N-terminus of GLP-2, to provide a N^(ter)-conjugate formof the peptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent isused based upon absolute peptide content. The mPEG-SPC reagent isweighed into a glass vial containing a magnetic stirrer bar. A solutionof GLP-2 prepared in phosphate buffered saline, PBS, pH 7.4 is added andthe mixture is stirred using a magnetic stirrer until the mPEG-SPC isfully dissolved. The stirring speed is reduced and the reaction isallowed to proceed to formation of conjugate product. The reaction isoptionally quenched to terminate the reaction. The pH of the conjugatesolution at the end of the reaction is measured and further acidified byaddition of 0.1M HCl, if necessary, to bring the pH of the finalsolution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-GLP-2conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) GLP-2-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of GLP-2, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected GLP-2(Prot-GLP-2, e.g.,Fmoc-His-Ala-Asp(tBu)-Gly-Ser(tBu)-Phe-Ser(tBu)-Asp(tBu)-Glu(tBu)-Met-Asn-Thr(tBu)-Ile-Leu-Asp(tBu)-Asn-Leu-Ala-Ala-Arg(Tos)-Asp(tBu)-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr(tBu)-Lys(Fmoc)-Ile-Tyr(tBu)-Asp(tBu)-OH)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 3-5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-GLP-2 is prepared in N,N-dimethylformamide is added and the mixtureis stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-GLP-2-N^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theGLP-2-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-GLP-2 Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock GLP-2 solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) GLP-2-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of GLP-2, to provide a Glu-conjugate form ofthe peptide. For coupling to the Glu residue, a protected GLP-2(Prot2-GLP-2, e.g.,Fmoc-His-Ala-Asp(tBu)-Gly-Ser(tBu)-Phe-Ser(tBu)-Asp(tBu)-Glu(OBz)-Met-Asn-Thr(tBu)-Ile-Leu-Asp(tBu)-Asn-Leu-Ala-Ala-Arg(Tos)-Asp(tBu)-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr(tBu)-Lys(Fmoc)-Ile-Tyr(tBu)-Asp(tBu)-O(tBu))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Glu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate forsubsequent coupling (Prot3-GLP-2, e.g.,Fmoc-His-Ala-Asp(tBu)-Gly-Ser(tBu)-Phe-Ser(tBu)-Asp(tBu)-Glu-Met-Asn-Thr(tBu)-Ile-Leu-Asp(tBu)-Asn-Leu-Ala-Ala-Arg(Tos)-Asp(tBu)-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr(tBu)-Lys(Fmoc)-Ile-Tyr(tBu)-Asp(tBu)-O(tBu))mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-GLP-2 is preparedin N,N-dimethylformamide is added and the mixture is stirred using amagnetic stirrer until the mPEG-NH₂ is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The conjugate solution is then analyzed by SDS-PAGEand RP-HPLC (C18) to determine the extent of Prot3-GLP-2-(Glu-β-mPEG)conjugate formation. The remaining protecting groups are removed understandard deprotection conditions to yield the GLP-2-Glu(O-mPEG)conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example 41 Gastrin-mPEG Conjugates

Gastrin is a hormone secreted by the G cells of the duodenum and in thepyloric antrum of the stomach which stimulate the secretion of gastricacid by the parietal cells of the stomach in response to stomachdistension, vagal stimulation, partially digested proteins, andhypercalcemia. Gastrin is a heptadecapeptide of the sequence,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH₂(PyrGPWLEEEEEA YGWMDF-NH₂, available from GenScript Corporation,Piscataway, N.J.; Cat. No. RP12740)

a) mPEG-N^(ter)-Gastrin—Via mPEG-SPC

Gastrin is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. An illustrativepolymeric reagent, mPEG-SPC reagent, is covalently attached to theN-terminus of Gastrin, to provide a N^(ter)-conjugate form of thepeptide. mPEG-SPC 20 kDa, stored at −20° C. under argon, is warmed toambient temperature. The reaction is performed at room temperature.About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used basedupon absolute peptide content. The mPEG-SPC reagent is weighed into aglass vial containing a magnetic stirrer bar. A solution of Gastrinprepared in phosphate buffered saline, PBS, pH 7.4 is added and themixture is stirred using a magnetic stirrer until the mPEG-SPC is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The reaction is optionallyquenched to terminate the reaction. The pH of the conjugate solution atthe end of the reaction is measured and further acidified by addition of0.1 M HCl, if necessary, to bring the pH of the final solution to about5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC(C18) to determine the extent of mPEG-N^(TEr)-Gastrin conjugateformation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Gastrin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of Gastrin, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protected Gastrin(Prot-Gastrin, e.g.,Fmoc-Glu(tBu)-Gly-Pro-Trp-Leu-Glu(tBu)-Glu(tBu)-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Tyr(tBu)-Gly-Trp-Met-Asp(tBu)-Phe-OH)is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. About3-5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-Gastrin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-Gastrin-C^(ter)-if/PEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Gastrin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) mPEG-N^(ter)-Gastrin Via mPEG-SMB

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock Gastrin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) Gastrin-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of Gastrin, to provide a Glu-conjugate formof the peptide. For coupling to the Glu residue, a protected Gastrin(Prot2-Gastrin, e.g.,Fmoc-Glu(OBz)-Gly-Pro-Trp-Leu-Glu(tBu)-Glu(tBu)-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Tyr(tBu)-Gly-Trp-Met-Asp(tBu)-Phe-NH₂) is prepared and purified according to standard automatedpeptide synthesis techniques known to those skilled in the art.Deprotection of the Glu(OBz) residue (H₂/Pd) yields the free-Glucarboxylate for subsequent coupling (Prot3-Gastrin, e.g.,Fmoc-Glu-Gly-Pro-Trp-Leu-Glu(tBu)-Glu(tBu)-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Tyr(tBu)-Gly-Trp-Met-Asp(tBu)-Phe-NH₂)mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. The reaction is performed at room temperature. A 5-foldmolar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Gastrin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Gastrin-(Glu-O-mPEG) conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theGastrin-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example KISS1 PEGylation of Kisspeptin-13 with mPEG-ButyrALD-30K(linear)

Kisspeptin-13 stock solution (KP-13; 0.454 mL of a 26.4 mg/mL stocksolution) in 50 mM sodium acetate, pH 4.0, and 2.907 mL of 50 mM sodiumacetate, pH 4.0, were mixed in a 50 mL polypropylene low endotoxinconical tube. PEG solution (three mol equivalents to the amount ofpeptide) was freshly prepared by dissolving 880 mg of linearmPEG-ButyrALD-30K PEG in 8.8 mL 50 mM sodium acetate, pH 4.0. Aftervigorous vortexing and 0.22 μm filtration, 8.292 mL of PEG solution wasadded drop-wise within 30 seconds to the peptide solution whilestirring. After 15 minutes, a freshly prepared solution of sodiumcyanoborohydride (0.347 mL of 50 mg/mL sodium cyanoborohydride inMilli-Q H₂O) was added (ten mol equivalents to PEG). The reactionmixture was allowed to gently stir at room temperature for 17 hours. Thereaction was diluted 1:5 with 10 mM sodium acetate, pH 4.0, and purifiedby cation exchange chromatography (HiTrap SP SEPHAROSE HP; 2×5 mLcolumns connected in series). Multiple loadings were necessary forpurification as the resin had a low binding capacity for the PEGylatedpeptide. A linear gradient (FIG. KISS1.1) separated the mono-conjugatefrom the non-conjugated peptide. Purification buffers were as follows:A: 10 mM sodium acetate, pH 4.0, and B: 10 mM sodium acetate, 1.0 Msodium chloride, pH 4.0. The diluted reaction mixture was loaded at 1mL/min with a two column volume wash after the load. The linear gradientconsisted of 0 to 60% B over ten column volumes at an elution flow rateof 1 mL/min. The purified mono-conjugate was determined to be 100% pureby reversed phase HPLC (FIG. KISS1.2 and T KISS1.1). MALDI-TOF analysis(FIG. KISS1.3), indicated the expected mass (34,017 Da) forKisspeptin-13 mono-PEGylated with a 30 kD PEG. Final conjugateconcentration was determined to be 5.17 mg/mL using a standard curve ofKisspeptin-13 with analytical RP-HPLC.

TIME (min) % B Flow rate (mL/min) 0.0 25 0.4 3.0 25 0.4 28.0 70 0.428.01 100 0.4 31.00 100 0.4 31.01 25 1.0 35.00 25 1.0T KISS1.1: Analytical RP-HPLC method. Symmetry C18, 3.5 μm, 3.6×75 mm.Mobile Phase A: 0.08% TFA/H₂O and B: 0.07% TFA/CH₃CN.

Example KISS2 PEGylation of Kisspeptin-10 (KP-10) with[mPEG-ButyrAldehyde-10K]

Stock solutions of 2.0 mg/mL KP-10 and 200 mg/mL mPEG-ButyrALD10K wereprepared in 2 mM HCl. To initiate a reaction, the two stock solutionsand a 1 M sodium acetate, pH 4.0, stock solution were brought to 25° C.,and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.0 mg/mL KP-10 (0.75 mM), 50 mM sodiumacetate and a 6-fold molar excess of mPEG-ButyrALD10K over KP-10. After15 minutes reaction, a 10-fold molar excess of NaBH₃CN over PEG wasadded and the reaction was allowed to continue for an additional 16hours at 25° C. After 16 hr 50 min total reaction time, the reaction wasquenched with 100 mM glycine in 100 mM HCl (10 mM final glycineconcentration) for 1 hour, after which glacial acetic acid was added toa final concentration of 5% (v/v).

The mono-PEGylated conjugate was purified from the reaction mixture byreversed phase chromatography using a column packed with CG71S media(Rohm Haas) on an AKTA Explorer 100 system (GE Healthcare). Buffer A was5% acetic acid/20% acetonitrile/75% H₂O (v/v), and Buffer B was 5%acetic acid/95% acetonitrile (v/v). The AKTA Explorer plumbing systemand the CG 71S resin were sanitized with 1 M HCl and 1 M NaOH and theresin was equilibrated with 10 column volumes Buffer A prior to sampleloading. After loading, the resin was washed with 6 CV of buffer A, andthe PEGylated and nonPEGylated peptides were eluted using a lineargradient from 100% A/0% B to 0% A/100% B over 15 column volume with alinear flow rate of 90 cm/hour.

Fractions collected during reversed phase chromatography with the CG71Sresin were analyzed using analytical reversed-phase HPLC, The mobilephases were: A, 0.08% TFA in water, and B, 0.05% TFA in acetonitrile. AWaters Symmetry C18 column (4.6 mm×75 mm) was used with a flow rate of0.5 ml/min and a column temperature of 60° C. Detection was carried outat 280 nm. The column was equilibrated in 25% B and conjugate separationwas achieved using the gradient timetable shown in T KISS2.1.

Step Time (min) % Mobile phase B 1 0.00 25.0 2 3.00 25.0 3 21.50 60.0 421.60 100.0 5 24.60 100.0 6 24.70 25.0

Fractions containing pure [mono]-[mPEG-ButyAldehyde-10K]-[Kisspeptin-10]as determined by analytical RP-HPLC were pooled, lyophilized and storedat −80° C.

A typical reversed phase CG71S chromatogram is shown in FIG. 2.1.RP-HPLC analysis of the purified conjugate is shown in FIG. 2.2, andMALDI-TOF analysis of the purified conjugate is shown in FIG. 2.3. Thepurity of the mono-PEG-conjugate was 98% by RP-HPLC analysis. The massas determined by MALDI-TOF was within the expected range.

Example KISS3 PEGylation of Kisspeptin-10 (KP-10) with[mPEG-ButyrAldehyde-30K]

Stock solutions of 2.0 mg/mL KP-10 and 200 mg/mL mPEG-butyrALD30K wereprepared in 2 mM HCl. To initiate a reaction, the two stock solutionsand a 1 M sodium acetate, pH 4.0, stock solution were brought to 25° C.,and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.0 mg/mL KP-10 (0.75 mM), 50 mM sodiumacetate and a 6-fold molar excess of mPEG-butyrALD30K over KP-10. After15 min reaction, a 10-fold molar excess of NaBH₃CN over PEG was addedand the reaction was allowed to continue for an additional 16 hours at25° C. After 16 hr 50 min total reaction time, the reaction was quenchedwith 100 mM glycine in 100 mM HCl (10 mM final glycine concentration)for 1 hour, after which glacial acetic acid was added to a finalconcentration of 5% (v/v).

The mono-PEGylated conjugate was purified from the reaction mixture byreversed phase chromatography using a column packed with CG71S media(Rohm Haas) on an AKTA Explorer 100 system (GE Healthcare). Buffer A was5% acetic acid/95% H₂O (v/v), and Buffer B was 5% acetic acid/95%acetonitrile (v/v). The AKTA Explorer plumbing system and the CG71Sresin were sanitized with 1 M HCl and 1 M NaOH and the resin wasequilibrated with 10 column volumes Buffer A prior to sample loading.After loading, the resin was washed with 6 CV of 80% Buffer A/20% BufferB and the PEGylated and nonPEGylated peptides were eluted using a lineargradient from 80% A/20% B to 40% A/60% B over 15 column volume with alinear flow rate of 90 cm/hour.

Fractions collected during reversed phase chromatography with the CG71Sresin were analyzed using reversed-phase HPLC. The mobile phases were:A, 0.08% TFA in water, and B, 0.05% TFA in acetonitrile. A WatersSymmetry C18 column (4.6 mm×75 mm) was used with a flow rate of 0.5ml/min and a column temperature of 60° C. Detection was carried out at280 nm. The column was equilibrated in 25% B and conjugate separationwas achieved using the gradient timetable shown in T KISS3.1

Step Time (min) % Mobile phase B 1 0.00 25.0 2 3.00 25.0 3 21.50 60.0 421.60 100.0 5 24.60 100.0 6 24.70 25.0

Fractions containing pure [mono]-[mPEG-ButyAldehyde30K]-[Kisspeptin-10]as determined by analytical RP-HPLC were pooled, lyophilized and storedat −80° C.

A typical reversed phase CG71S chromatogram is shown in FIG. KISS3.1.RP-HPLC analysis of the purified conjugate is shown in FIG. KISS3.2, andMALDI-TOF analysis of the purified conjugate is shown in FIG. KISS3.3.The purity of the mono-PEG-conjugate was 99.2% by RP-HPLC analysis. Themass as determined by MALDI-TOF was within the expected range. The peakat 33.5 kDa is within the expected range for the molecular weight of themono-PEG-conjugate. The peak at 66.1 kDa may represent the singlecharged mono-[mPEG-Butyraldehyde-30K]-[Kisspeptin-10] dimer formedduring MALDI-TOF analysis.

Example KISS4 PEGylation of Kisspeptin-10 (KP-10) with[mPEG2-CAC-FMOC-NHS-40K]

Stock solutions of 2.0 mg/mL KP-10 and 200 mg/mL mPEG2-CAC-FMOC-NHS-40Kwere prepared in 2 mM HCl. To initiate a reaction, the two stocksolutions and a 1 M MES, pH 6.0, stock solution were brought to 25° C.,and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.0 mg/mL KP-10 (0.75 mM), 50 mM MES and a6-fold molar excess of mPEG-butyrALD30K over KP-10. The reaction wasallowed to proceed for 2.5 hours at 25° C. After 2.5 hr, the reactionwas quenched with 100 mM glycine in 100 mM HCl (10 mM final glycineconcentration) for 10 minutes, after which glacial acetic acid was addedto a final concentration of 5% (v/v).

The mono-PEGylated conjugate was purified from the reaction mixture byreversed phase chromatography using a column packed with CG71S media(Roam Haas) on an AKTA Explorer 100 system (GE Healthcare). Buffer A was5% acetic acid/95% H₂O (v/v), Buffer B1 was 5% acetic acid/95% ethanol(v/v), and Buffer B2 was 5% acetic acid/95% acetonitrile (v/v). The AKTAExplorer plumbing system and CG71S were sanitized with 1 M HCl and 1 MNaOH and the resin was equilibrated with 10 column volumes Buffer Aprior to sample loading. After loading, unreacted PEG reagent was elutedwith a linear gradient from 100% A/0% B1 to 0% A/100% B1 over 10 columnvolumes with a linear flow rate of 90 cm/hour, followed by a 100% BufferA wash over 4 column volumes. The PEGylated and nonPEGylated peptideswere eluted using a linear gradient from 100% A/0% B2 to 40% A/60% B2over 15 column volumes with a linear flow rate of 90 cm/hour.

Fractions collected during reversed phase chromatography with the CG71Sresin were analyzed using analytical reversed-phase HPLC. The mobilephases were: A, 0.08% TFA in water, and B, 0.05% TFA in acetonitrile. AWaters Symmetry C18 column (4.6 mm×75 mm) was used with a flow rate of0.5 ml/min and a column temperature of 60° C. Detection was carried outat 280 nm. The column was equilibrated in 25% B and conjugate separationwas achieved using the gradient timetable shown in T KISS4.1.

Step Time (min) % Mobile phase B 1 0.00 25.0 2 3.00 25.0 3 21.50 60.0 421.60 100.0 5 24.60 100.0 6 24.70 25.0

Fractions containing pure mono-[mPEG2-CAC-FMOC-40K]-[Kisspeptin-10] asdetermined by RP-HPLC were pooled, lyophilized and stored at −80° C.

A typical reversed phase CG71S chromatogram is shown in FIG. KISS4.1.RP-HPLC analysis of the purified conjugate is shown in FIG. KISS4.3, andMALDI-TOF analysis of the purified conjugate is shown in FIG. KISS4.3.The purity of the mono-PEG-conjugate was 99.6% by RP-HPLC analysis. Themass as determined by MALDI-TOF was within the expected range.

Example KISS5 PEGylation of Kisspeptin-10 (KP-10) withN-m-PEG-Benzamide-p-Succinimidyl Carbonate (SBC)-30K

A stock solution of 2.0 mg/mL KP-10 was prepared in 2 mM HCl. Toinitiate a reaction, the KP-10 stock solution was brought to 25° C., a15-fold molar excess of SBC-30K lyophilized powder was added withstirring followed immediately with the addition of 1 M MES, pH 6, togive final concentrations of 1.0 mg/mL KP10 (0.75 mM) and 50 mM MES. Thereaction was allowed to proceed for 20 minutes at 25° C. After 20 min,the reaction was quenched with 100 mM glycine in 100 mM HCl (10 mM finalglycine concentration) for 10 minutes, after which glacial acetic acidwas added to a final concentration of 5% (v/v).

The mono-PEGylated conjugate was purified from the reaction mixture byreversed phase chromatography using a column packed with CG71S media(Rohm Haas) on an AKTA Explorer 100 system (GE Healthcare). Buffer A was5% acetic acid/95% H₂O (v/v), Buffer B1 was 5% acetic acid/95% ethanol(v/v), and Buffer B2 was 5% acetic acid/95% acetonitrile (v/v). The AKTAExplorer plumbing system and the CG71S resin were sanitized with 1 M HCland 1 M NaOH and the resin was equilibrated with 10 column volumesBuffer A prior to sample loading. After loading, unreacted PEG reagentwas eluted with a linear gradient from 100% A/0% B1 to 0% A/100% B1 over10 column volumes with a linear flow rate of 90 cm/hour, followed by a100% A wash over 4 column volumes. The PEGylated and nonPEGylatedpeptides were eluted using a linear gradient from 100% A/0% B2 to 40%A/60% B2 over 15 column volumes with a linear flow rate of 90 cm/hour.

Fractions collected during reversed phase chromatography with the CG71Sresin were analyzed using analytical reversed-phase HPLC. The mobilephases were: A, 0.08% TFA in water, and B, 0.05% TFA in acetonitrile. AWaters Symmetry C18 column (4.6 mm×75 mm) was used with a flow rate of0.5 ml/min and a column temperature of 60° C. Detection was carried outat 280 nm. The column was equilibrated in 25% B and conjugate separationwas achieved using the gradient timetable shown in T KISS5.1.

Step Time (min) % Mobile phase B 1 0.00 25.0 2 3.00 25.0 3 21.50 60.0 421.60 100.0 5 24.60 100.0 6 24.70 25.0

Fractions containing pure mono-[mPEG-SBC-30K]-[Kisspeptin-10] asdetermined by RP-HPLC were pooled, lyophilized and stored at −80° C. Atypical reversed phase CG71S chromatogram is shown in FIG. KISS5.1.SDS-PAGE analysis of purified mono-[mPEG-SBC-30K]-[Kisspeptin-10] isshown in FIG. KISS5.2. RP-HPLC analysis of the purified conjugate isshown in FIG. KISS5.3, and MALDI-TOF analysis of the purified conjugateis shown in FIG. KISS5.4. The purity of the mono-PEG-conjugate was >95%by SDS-PAGE and 95.4% by RP-HPLC analysis. The mass as determined byMALDI-TOF was within the expected range.

Example KISS6 PEGylation of Kisspeptin-54 (KP-54) withmPEG2-ButyrAldehyde-40K

Stock solutions of 2.0 mg/mL KP-54 and 200 mg/mL mPEG-butyrALD40K wereprepared in 2 mM HCl. To initiate a reaction, the two stock solutionsand a 1 M MES, pH 6.0, stock solution were brought to 25° C., and thethree stock solutions were mixed (PEG reagent added last) to give finalconcentrations of 1.0 mg/mL KP-54 (0.15 mM), 50 mM MES and a 6-foldmolar excess of mPEG-butyrALD40K over KP-54. After 15 min reaction, a10-fold molar excess of NaBH₃CN over PEG was added and the reaction wasallowed to continue for an additional 16 hours at 25° C. After 16 hr 15min total reaction time, the reaction was quenched with 100 mM glycinein 100 mM HCl (10 mM final glycine concentration) for 10 minutes. Thereaction mixture was diluted with sterile deionized H₂O until theconductivity was below 1.0 mS/cm and the pH was then adjusted to 6.0with 1 M Na₂CO₃/NaHCO₃, pH 10.0.

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using SPHP media (GE Healthcare) on anAKTA Explorer 100 system (GE Healthcare). Buffer A was 20 mM MES, pH6.0, Buffer B was 20 mM MES and 1 M NaCl, pH 6.0. The AKTA Explorerplumbing system and SPHP resin were sanitized with 1 M HCl and 1 M NaOHand the SPHP resin was equilibrated with 10 column volumes Buffer Aprior to sample loading. After loading and a column wash with 5 columnvolumes Buffer A, the PEGylated and nonPEGylated peptides were elutedusing a linear gradient from 100% A/0% B to 0% A/100% B over 15 columnvolumes with a linear flow rate of 90 cm/hour.

Fractions collected during cation exchange chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.08%TFA in water, and B, 0.05% TFA in acetonitrile. A Waters Symmetry C18column (4.6 mm×75 mm) was used with a flow rate of 0.5 ml/min and acolumn temperature of 60° C. Detection was carried out at 280 nm. Thecolumn was equilibrated in 25% B and conjugate separation was achievedusing the gradient timetable shown in T KISS6.1.

Step Time (min) % Mobile phase B 1 0.00 25.0 2 3.00 25.0 3 21.50 60.0 421.60 100.0 5 24.60 100.0 6 24.70 25.0

Fractions containing pure mono-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54]as determined by RP-HPLC were pooled and concentrated over a reversedphase CG71S column. The column was washed with 5% acetic acid inacetonitrile and equilibrated with 5% acetic acid prior to loading.After loading, the column was washed with 5% acetic acid and thePEGylated peptide was eluted with a linear gradient from 5% acetic acidto 5% acetic acid/95% acetonitrile (v/v) over 5 column volumes.Fractions containing the conjugate were pooled, lyophilized and storedat −80° C.

A typical cation exchange SPHP chromatogram is shown in FIG. KISS6.1.SDS-PAGE analysis of purifiedmono-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54] is shown in FIG. KISS6.2.RP-HPLC analysis of the purified conjugate is shown in FIG. KISS6.3, andMALDI-TOF analysis of the purified conjugate is shown in FIG. KISS6.4.

The purity of the mono-PEG-conjugate was >95% by SDS-PAGE and 100% byRP-HPLC analysis. The mass as determined by MALDI-TOF was within theexpected range. The major peak at 49 kDa is within the expected rangefor the molecular weight of[mono]-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54]. The peak at 24 kDarepresents the double charged conjugate and the peak at 97 kDa mayrepresent the single charged conjugate dimer formed during MALDI-TOFanalysis.

Example KISS7 a) mPEG-N^(ter)-KISS1 Via mPEG-SPC

KISS1 is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of KISS1, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. An X-fold molar excess of mPEG-SPC 20 kDareagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of KISS1 prepared in phosphate buffered saline, PBS, pH 7.4is added and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-KISS1conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) KISS1-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of KISS1, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected KISS1(Prot-KISS1, e.g,Fmoc-Ile-Pro-Cys(tBu)-Asn-Asn-Lys(Fmoc)-Gly-Ala-His-Ser(Dmab)-Val-Gly-Leu-Met-Trp-Trp-Met-Leu-Ala-Arg(Tos))is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. A X-fold molar excess ofmPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-KISS1 is prepared in N,N-dimethylformamide is added and the mixtureis stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-KISS1-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theKISS1-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) KISS1-Cys(S-mPEG)

mPEG-Maleimide is obtained having a molecular weight of 5 kDa and havingthe basic structure shown below:

mPEG-MAL, 5 kDa

KISS1, which has a thiol-containing cysteine residue, is dissolved inbuffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-1″-KISS1 Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock KISS1 solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

d) KISS1-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of KISS1, to provide a Glu-conjugate form ofthe peptide. For coupling to the Glu residue, a protected KISS1(Prot2-KISS1) is prepared and purified according to standard automatedpeptide synthesis techniques known to those skilled in the art.Deprotection of the Glu(OBz) residue (H₂/Pd) yields the free-Glucarboxylate for subsequent coupling (Prot3-KISS1) mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. A 5-fold molar excess ofmPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt3-KISS1 is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-KISS1-(Glu-O-mPEG) conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theKISS1-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

Example KISS8

A FLIPR assay was conducted to screen Kisspeptin and PEG-Kisspeptinpeptides for dose-dependent agonist activities on the GPR54 G-Proteincoupled receptor. EC₅₀ potency values were determined for each compoundon the GPR54 GPCR, and Metastin 45-54 (Kisspeptin 10) was used as thereference agonist.

Sample preparation: Sample compounds are listed in T KISS8.1. Prior toassay, CAC-PEG2-FMOC-NHS-40K-Kisspeptin 10 andmono-mPEG-SBC-30K-Kisspeptin 10 (provided in 2 mM HCl) were diluted 1:1in 200 mM or 10 mM HEPES buffer, pH 7, respectively, and incubated at37° C. for 0, 24, 48, and 96 h for CAC-PEG2-FMOC-NHS-40K-Kisspeptin 10;0, and 2 h for mono-mPEG-SBC-30K-Kisspeptin 10). All compounds werediluted in their storage solvents to produce 250× (of the top doselisted below) master stock solutions. Compounds were then transferredfrom their master stock solutions into a daughter plate that was used inthe assay. Each 250× solution was diluted into assay buffer (1×HBSS with20 mM HEPES and 2.5 mM Probenecid) to obtain the final top testconcentration.

Calcium flux agonist assay: Chemicon's cloned human GPR54-expressingcell line is made in the Chem-1 host, which supports high levels ofrecombinant GPR54 expression on the cell surface and contains highlevels of the promiscuous G protein Gα15 to couple the receptor to thecalcium signaling pathway. Sample compounds were plated in aneight-point, four-fold serial dilution series with a top concentrationof 0.375 μM (except for CAC-PEG2-FMOC-NHS-40K-Kisspeptin 10, topconcentration of 1.25 μM). Reference agonist was handled as mentionedabove, serving as assay control. Assay was read for 180 seconds usingthe FLIPR^(TETRA). All plates were subjected to appropriate baselinecorrections. Once baseline corrections were processed, maximumfluorescence values were exported and data manipulated to calculatepercentage activation and Z′. Dose response curves were generated usingGraphPad Prism. The curves were fit by utilizing sigmoidal dose response(variable slope) fitting with the bottom parameter fixed at 0 (FIG.KISS8.1-KISS8.3).

TABLE KISS8.1 Dose half-life of response stable releasable release topdose KP10 n/a 0.375 μM mPEG-ALD10K-KP10 n/a 0.375 μM mPEG-ALD30K-KP10n/a 0.375 μM CAC-PEG2-Fmoc-NHS- 32 h  1.25 μM 40K-KP10 mPEG-SBC30K-KP1027 min 0.375 μM KP13 n/a 0.375 μM mPEG-ALD30K-KP13 n/a 0.375 μM KP54 n/a0.375 μM mPEG-ALD40K-KP54 n/a 0.375 μMFIG. KISS8.1. Agonist activity at GPR54 for stable PEG conjugates ofKisspeptin 10, Kisspeptin 13, and Kisspeptin 54.FIG. KISS8.2. Agonist activity at GPR54 for releasable PEG conjugate ofKisspeptin 10.FIG. KISS8.3. Agonist activity at GPR54 for releasable PEG conjugate ofKisspeptin 10.

TABLE KISS8.2 Summary of EC₅₀ values of agonist activation at GPR54.Time of Fold change compared compound release EC₅₀ (nM) to metastin KP10n/a 10 1 Ald10K-KP10 No activity — Ald30K-KP10 No activity —SBC-30K-KP10  0 h 280 23 SBC-30K-KP10  2 h 200 17 CAC-40K-KP10  0 h 1700155 CAC-40K-KP10 24 h 120 9 CAC-40K-KP10 48 h 74 6 CAC-40K-KP10 96 h 474 KP13 n/a 11 1 Ald30K-KP13 No activity — KP54 190 16 Ald40K-KP54 Noactivity — Metastin (cntl) 10-14* — *varied depending on the individualtest plate (samples received in different buffers were tested againstmetastin control in the same buffer)

Stable PEG conjugates of Kisspeptin 10, Kisspeptin 13, and Kisspeptin 54do not retain agonist activity at the GPR54 receptor, whereas bothKisspeptin 10 releasable conjugates show partial activity after releasein buffer at pH 7.0 (T KISS8.2). The SBC-30K Kisspeptin 10 conjugate hasa half-life release rate of 27 minutes, and the activity at Oh and after2 h of release were similar, EC₅₀=280 and 200 nM, respectively, about23- and 17-fold less than the metastin control. The activity exhibitedby SBC-30K Kisspeptin 10 (0 hr) is believed to be due to release of thepeptide from the conjugate prior to assay. The CAC-40K Kisspeptin 10conjugate, with a half-life of release of 32 h, had EC₅₀ values of 1600,120, 74, and 47 nM after 0, 24, 48, and 96 h release, and showed155-fold, 9-fold, 6-fold, and 4-fold less activity compared to metastinafter 0, 24, 48, and 96 h release, respectively. We did not test theactivity of Kisspeptin 10 (metastin) after incubation at 37° C. for anequivalent time.

E ZIC1

Ziconotide conjutgate strategy: The N-terminal amine and four s-aminegroups on lysine residues are the targeted positions for PEGylation. Thechemistry of ziconotide PEGylation with the non-releasable mSBA-30K PEGreagent is illustrated.

PEGylation of a drug with a mSBA-NHS reagent.

PEGylation with releasable PEG reagents such as phenyl carbamate arealso performed. Figure shows the PEGylation of ziconotide with areleasable mSBC-30K PEG reagent and the potential pathway to regeneratethe parent drug from the conjugate.

Example of the formation of a carbamate PEG drug conjugate and apossible pathway of regenerating the parent drug under physiologicalconditions.

PEGylation with releasable PEG reagents such as fluorenylmethylchloroformate (FMOC) are also performed. Figure below shows thePEGylations of zinconotide with releasable C2-20K-FMOC and CAC-40K-FMOCPEG reagents and the potential pathways to regenerate the parent drugfrom the conjugates. By fine tuning the PEG reagent structures, the PEGrelease rate from the conjugate parent drug can be altered.

Example of the formation of a C2-FMOC-PEG drug conjugate and a possiblepathway of regenerating the parent drug under physiological conditions.

Example of the formation of a CAC-FMOC-PEG drug conjugate and a possiblepathway of regenerating the parent drug under physiological conditions.

E ZIC2 PEGylation of Ziconotide with mPEG-C2-FMOC-20K-NHS

mPEG-C2-FMOC-20K-NHS

mono-mPEG-C2-FMOC-20K-ziconotide was produced in a 2.4-mL reactionmixture consisting of 0.44 mL water, 0.096 mL 0.5 M HEPES, pH 7.4, 0.12mL of 100 mg/ml ziconotide and 2.14 ml of 100 mg/mL mPEG-C2-FMOC-20K.The molar ratio between ziconotide and PEG reagent was 1:2 after thecorrection of purity of the PEG reagent. mPEG-C2-FMOC-20K, the lastreagent added to the mixture, was dissolved in 2 mM HCl to a finalconcentration of 100 mg/mL immediately before addition. The dissolvedPEG reagent was added to the reaction mixture with stirring. Thereaction mixture was incubated at 25° C. with stirring for 45 minutes.After 45 minutes, 0.126 mL 0.2 M glycine (unbuffered) was added into thereaction mixture to quench the unreacted PEG reagent. After anadditional 30 minutes of stirring at 25° C., the pH of the reactionmixture was adjusted to 5.0 at room temperature with acetic acid. Thereaction mixture was diluted 1:10 with 20 mM sodium acetate, pH 5.0, andpurified by cation exchange chromatography (HiTrap SP Sepharose HP; 5mL). A linear salt gradient (FIG. ZIC2.1) separated the mono-conjugatefrom the di- and high PEGylated products and unrereacted peptide.Purification buffers were as follows: A: 20 mM sodium acetate, pH 5.0,and B: 20 mM sodium acetate, 1.0 M sodium chloride, pH 5.0. The dilutedreaction mixture was loaded at 0.4 mL/min with a two column volume washafter the load. The linear gradient consisted of 0 to 60% B over twentycolumn volumes at an elution flow rate of 0.4 mL/min. The purifiedmono-conjugate was determined to be 98% pure by reversed phase HPLC(FIG. ZIC2.2 and T ZIC2.1). MALDI-TOF analysis indicated the expectedmass (23.9 kDa) for ziconotide mono-PEGylated with a 20 kDa PEG (FIG.ZIC2.3). The final conjugate concentration was determined to be 0.21mg/mL using a standard curve of ziconotide with the BCA assay.

TABLE ZIC2.1 Analytical RP-HPLC method: Poroshell, 5 μm, 2.1 × 75 mm.Mobile Phase A: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN TIME (min) % B Flowrate (mL/min) 0.0 0 0.5 1.0 0 0.5 10 80 0.5 10.1 95 0.5 12.1 95 0.5 12.20 0.5 16.0 0 0.5

E ZIC3 PEGylation of Ziconotide with mPEG-CAC-FMOC-40K-NHS

mPEG-CAC-FMOC-40K-NHS

mono-mPEG-CAC-FMOC-40K-ziconotide was produced in a 4.8-mL reactionmixture consisting of 2.32 mL water, 0.192 mL 0.5 M HEPES, pH 7.4, 0.12mL of 100 mg/ml ziconotide and 2.16 ml of 100 mg/mL mPEG-CAC-FMOC-40K.The molar ratio between ziconotide and PEG reagent was 1:1 after thecorrection of purity of the PEG reagent. mPEG-CAC-FMOC-40K, the lastreagent added to the mixture, was dissolved in 2 mM HCl to a finalconcentration of 100 mg/mL immediately before addition. The dissolvedPEG reagent was added to the reaction mixture with stirring. Thereaction mixture was incubated at 25° C. with stirring for one hour.After one hour, 0.252 mL 0.2 M glycine (unbuffered) was added into thereaction mixture to quench the unreacted PEG reagent. After anadditional 30 minutes of stirring at 25° C., the pH of the reactionmixture was adjusted to 5.0 at room temperature with acetic acid. Thereaction mixture was diluted 1:10 with 10 mM sodium acetate, pH 5.0, andpurified by cation exchange chromatography (HiTrap SP Sepharose HP; 5mL). A linear salt gradient (FIG. ZIC3.1) separated the mono-conjugatefrom the di- and high PEGylated products and unrereacted peptide.Purification buffers were as follows: A: 10 mM sodium acetate, pH 5.0,and B: 10 mM sodium acetate, 1.0 M sodium chloride, pH 5.0. The dilutedreaction mixture was loaded at 0.4 mL/min with a five column volume washafter the load. The linear gradient consisted of 0 to 60% B over twentycolumn volumes at an elution flow rate of 0.4 mL/min. The purifiedmono-conjugate was determined to be 93% pure by reversed phase HPLC(FIG. ZIC3.2 and T ZIC3.1). MALDI-TOF analysis indicated the expectedmass (44.5 kDa) for ziconotide mono-PEGylated with a 40 kDa PEG (FIG.ZIC3.3). Final conjugate concentration was determined to be 0.17 mg/mLusing a standard curve of ziconotide with the BCA assay.

E ZIC4

PEGylation of Ziconotide with mPEG-SBA-30K-NHS

mPEG-SBA-30K-NHS

mono-mPEG-C2-FMOC-20K-ziconotide was produced in a 6.0-mL reactionmixture consisting of 4.27 mL water, 0.24 mL 0.5 M HEPES, pH 7.4, 0.12mL of 100 mg/ml ziconotide and 1.36 ml of 100 mg/mL mPEG-SBA-30K. Themolar ratio between ziconotide and PEG reagent was 1:2 after thecorrection of purity of the PEG reagent. mPEG-SBA-30K, the last reagentadded to the mixture, was dissolved in 2 mM HCl to a final concentrationof 100 mg/mL immediately before addition. The dissolved PEG reagent wasadded to the reaction mixture with stirring. The reaction mixture wasincubated at 25° C. with stirring for one hour. After one hour, 0.315 mL0.2 M glycine (unbuffered) was added into the reaction mixture to quenchthe unreacted PEG reagent. After an additional 30 minutes of stirring at25° C., the pH of the reaction mixture was adjusted to 5.0 at roomtemperature with acetic acid. The reaction mixture was diluted 1:10 with10 mM sodium acetate, pH 5.0, and purified by cation exchangechromatography (HiTrap SP Sepharose HP; 5 mL). A linear salt gradient(FIG. ZIC4.1) separated the mono-conjugate from the di- and highPEGylated products and unrereacted peptide. Purification buffers were asfollows: A: 10 mM sodium acetate, pH 5.0, and B: 10 mM sodium acetate,1.0 M sodium chloride, pH 5.0. The diluted reaction mixture was loadedat 0.4 mL/min with a five column volume wash after the load. The lineargradient consisted of 0 to 60% B over twenty column volumes at anelution flow rate of 0.4 mL/min. The purified mono-conjugate wasdetermined to be 97% pure by reversed phase HPLC (FIG. ZIC4.2 and TZIC4.1). MALDI-TOF analysis indicated the expected mass (34.2 kDa) forziconotide mono-PEGylated with a 30 kDa PEG (FIG. ZIC4.3). Finalconjugate concentration was determined to be 0.13 mg/mL using a standardcurve of ziconotide with the BCA assay.

E ZIC5 PEGylation of Ziconotide with mPEG-SBC-30K-NHS

mPEG-SBC-30K-NHS

mono-mPEG-SBC-30K-ziconotide was produced in a 0.5-mL reaction mixtureconsisting of 0.47 mL water, 0.02 mL 0.5 M HEPES, pH 7.4, and 0.01 mL of100 mg/ml ziconotide. With stirring, 23.6 mg of solid mPEG-SBC-30K-NHSwas added. 10 minutes after addition of the PEG reagent, the pH of thereaction mixture was adjusted to 5.0 with 6.2 μL of 1M acetic acid. Thereaction mixture was diluted 1:10 with 10 mM sodium acetate, pH 5.0, andpurified by cation exchange chromatography (HiTrap SP Sepharose HP; 1mL). A linear salt gradient (FIG. ZIC5.1) separated the mono-conjugatefrom the di- and high PEGylated products and unreacted peptide.Purification buffers were as follows: A: 10 mM sodium acetate, pH 5.0,and B: 10 mM sodium acetate, 1.0 M sodium chloride, pH 5.0. The dilutedreaction mixture was loaded at 0.4 mL/min with a two column volume washafter the load. The linear gradient consisted of 0 to 100% B over twentycolumn volumes at an elution flow rate of 0.4 mL/min. Five peaks wereobserved in the cation exchange chromatogram (FIG. ZIC5.1). Based onSDS-PAGE analysis of aliquots collected from peaks 1 and 5, peak 1corresponds to the unreacted PEG reagent and highly PEGylated ziconotideand peak 5 corresponds to unreacted ziconotide. Based on the peakretention times during FPLC chromatography, we speculate that peaks 2and 3 correspond to different positional isomers ofmono-PEGylated-ziconotide and peak 4 corresponds to tagged ziconotide inwhich the PEG group(s) have been released from the peptide. The FPLC andsubsequent analytical results strongly suggest that the SBC-ziconotideconjugate is very unstable.

E ZIC6 N-Type Calcium Channel Binding Assay

Competition binding experiments are conducted by incubating membraneswith 0.01 nM of radioligand, [¹²⁵I] ω-conotoxin GVIA, in the presence ofvariable concentrations (0.3 μM to 30 nM) of test compounds. Thereaction is carried out in 50 mM HEPES (pH 7.4) containing 0.2% BSA at25° C. for 1 hour. Following incubations, the membranes are washed, andthe bound radioactivity is measured. Non-specific binding is measured inthe presence of 0.1 μM ω-conotoxin GVIA as the cold ligand; this valueis subtracted from the total binding to yield the specific binding ateach test compound concentration.

IC₅₀ values are obtained from non-linear regression analysis ofdose-response curves (FIG. ZIC6.1) and are calculated for thosecompounds that showed >50% inhibition of binding at the highestconcentration tested. K_(i) is obtained using the Cheng Prusoffcorrection using experimental K_(d) values that are previouslydetermined under these assay conditions.

TABLE ZIC6.1 Summary of binding affinity. Fold Change Relative toCompound MW (Da) Ki (nM) Parent Ziconotide 2,639 0.029 1Mono-mPEG-20K-C2-FMOC- 23,900 0.543 19 ZiconotideMono-mPEG-30K-SBA-Ziconotide 34,200 0.707 24 Mono-mPEG-40K-CAC-FMOC-44,500 0.676 23 Ziconotide

TABLE 2 Compounds. Stock concentration based on peptide PEG Release rate(if Compound PEG (mg/mL) Storage buffer applicable) Ziconotide — 100Water — Mono-mPEG-20K- Releasable 0.21 Na-acetate: 20 mM, 55% after 24 hand 85% C2-FMOC- NaCl: after 42 h @ 37° C. in Ziconotide 150 mM, pH PBSat pH 7.38 5.0 Mono-mPEG-30K- Stable 0.13 Na-acetate: 10 mM, —SBA-Ziconotide NaCl: 150 mM, pH 5.0 Mono-mPEG-40K- Releasable 0.17Na-acetate: 10 mM, 15% after 24.5 h and CAC-FMOC- NaCl: 22% after 42.5 h@ 37° C. Ziconotide 150 mM, pH in PBS at pH 7.38 5.0

E BIP1 Biphalin-mPEG Conjugates

a) mPEG-N^(ter)-Biphalin Via mPEG-SPC

Biphalin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of biphalin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 5-fold molar excess of mPEG-SPC 20kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of biphalin prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-biphalin conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Biphalin-Cys(S-mPEG)

mPEG-Maleimide is obtained having a molecular weight of 5 kDa and havingthe basic structure shown below:

mPEG-MAL, 5 kDa

Biphalin, which is modified to contain a thiol-containing cysteineresidue, is dissolved in buffer. To this peptide solution is added a 3-5fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at roomtemperature under an inert atmosphere for several hours. Analysis of thereaction mixture reveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

c) mPEG-N^(ter)-Biphalin Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

(mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock biphalin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

E BIP2 PEGylation of Biphalin with mPEG-SPA-2K

The conjugation reaction took place in acetonitrile. 10.7 mg biphalinwas first dissolved into 7.6 mL acetonitrile followed by the addition of8.1 μL triethylamine. 154 mg SPA-2K was dissolved into 7.6 mLacetonitrile. To start the conjugation reaction, 2.53 mL SPA-2K solutionwas added to 7.6 mL biphalin solution drop by drop under rapid stirring.The SPA-2K to biphalin molar ratio was 2.4 with SPA-2K in excess. Thereaction was allowed to proceed for 66 h at 21° C. for completion. Theformation of (SPA-2K)₂-biphalin was confirmed by analytical RP-HPLC (TBIP2).

TABLE BIP 2.1 Analytical RP-HPLC method. Column: Waters Xbridge C18 5 μm4.6 × 160 mm. Mobile Phase A: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN. Columntemperature: 40° C. UV_(280 nm) is used to follow the elution. TIME(min) % B Flow rate (mL/min) 0.0 20 1 5 30 1 35 60 1 40 80 1 41 20 1

The (SPA-2K)₂-biphalin was purified by a CG-71S reverse phase resinusing an AKTA Basic System. The reaction mixture was first diluted 5fold with solvent A [0.1% TFA in water] to reduce sample viscosity. Thediluted sample mixture was then loaded onto the CG-71S column at a flowrate of 10 mL/min. After sample loading, the column was first washedwith 2 CV solvent A. This was followed by 2 CV 30% solvent B [SolventB=0.1% TFA in acetonitrile] wash. A gradient elution was next appliedfrom 30 to 45% solvent B in 15 CV. The (SPA-2K)₂-biphalin was eluted inthis step. The column was finally washed with 1 CV 80% solvent B. Theflow rate was constant at 10.25 ml/min throughout the purificationprocess. The chromatogram of the loading and elution is shown in FIG.BIP2.1.

FIG. BIP2.1: (SPA-2K)₂-biphalin purification with CG-71S resin. TheUV_(280nm) absorption curve and the solvent B percentage are shown.

The CG-71S column peak fractions were analyzed by the analytical RP-HPLCmethod (FIG. BIP2.2). Based on their high purities, fractions 32 to 40(across the whole (SPA-2K)₂-biphalin peak) were pooled.

This purified (SPA-2K)₂-biphalin pool was lyophilized to removeacetonitrile. The lyophilized pellet was reconstituted into 4 mL 20 mMacetate buffer, pH 4.0. The biphalin concentration in the reconstituted(SPA-2K)₂-biphalin was measured to be 0.92 mg/mL by BCA. The purity wasdetermined at 95.5% by RP-HPLC (FIG. BIP2.2). The number-averagemolecular weight was calculated to be 5279.15 Da by MALDI-TOF MS (FIG.BIP2.3). A final yield of 2.8 mg purified (SPA-2K)₂-biphalin wasobtained.

E BIP3 PEGylation of Biphalin with 2,7-C2-PEG2-FMOC-NHS-20K

The conjugation reaction took place in an aqueous environment. 18 mgbiphalin was first dissolved into 10 mL PBS buffer to make a 1.8 mg/mLstock solution. 800 mg C2-20K was dissolved into 8 mL 2 mM HCl to make a100 mg/mL stock solution. To initiate the conjugation, 7.5 mL C2-20Kstock solution was slowly mixed into 8.9 mL biphalin stock solution dropby drop under rapid stirring. 8.9 mL 10×PBS buffer was added into thereaction mixture to maintain a relatively neutral pH during the reaction(measured at 6.8). The C2-20K to biphalin molar ratio was 3.0 withC2-20K in excess. The reaction was allowed to proceed for 180 min at 21°C. The formation of (C2-20K)₂-biphalin was confirmed by analyticalRP-HPLC.

TABLE BIP3.1 Analytical RP-HPLC method used to monitor(C2-20K)₂-biphalin production. Column: Waters Xbridge C18 5 μm 4.6 × 160mm. Mobile Phase A: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN. Columntemperature: 40° C. UV_(280 nm) was used to follow the elution. TIME(min) % Mobile phase B Flow rate (mL/min) 0.0 20 1.0 5 30 1.0 35 60 1.040 80 1.0 41 20 1.0

The (C2-20K)₂-biphalin was purified by a CG-71S reverse phase resinusing an AKTA Basic System. The reaction mixture was first diluted 5fold with solvent A [0.1% TFA in water] to reduce sample viscosity. Thediluted sample mixture was loaded onto the CG-71S column at 10 mL/min.After sample loading, the column was first washed with 2 CV 10% solventB. This was followed by 3 CV 30% solvent B washing. Peaks I was elutedin this step. A linear gradient elution of 30 to 45% solvent B was nextapplied within 15 CV. Peak II was eluted in this step. The flow rate wasconstant at 10.25 ml/min throughout the purification process. Thechromatogram of the loading and elution is shown in FIG. BIP3.1.

The CG-71S column peak I and II fractions were analyzed by theanalytical RP-HPLC method. The RP-HPLC data indicated that mostcontaminants (free PEG and (C2-20K)₁-biphalin) were washed off in peakI. The desired (C2-20K)₂-biphalin was eluted in peak II. Based on theirpurities, fractions 28 to 44 in peak II were pooled.

The purified (C2-20K)₂-biphalin pool was lyophilized to removeacetonitrile. The lyophilized pellet was reconstituted into 8 mL 20 mMacetate buffer, pH 4.0. The biphalin concentration in the reconstituted(C2-20K)₂-biphalin was measured to be 0.99 mg/mL by BCA. The purity wasdetermined at 97.9% by RP-HPLC (FIG. BIP3.2). The number-averagemolecular weight was calculated to be 42055.99 Da by MALDI-TOF (FIG.BIP3.3). A final yield of 7.43 mg purified (C2-20K)₂-biphalin wasobtained.

E BIP4 PEGylation of Biphalin with 4,7-CAC-PEG2-FMOC-NHS-20K

4,7-CAC-PEG2-FMOC-NHS-20K (CAC-20K)

The conjugation reaction took place in an aqueous environment. 8 mgbiphalin was first dissolved into 4.4 mL PBS buffer to make a 1.8 mg/mLstock solution. 650 mg CAC-20K was dissolved into 6.5 mL 2 mM HCl tomake a 100 mg/mL stock solution. To initiate the conjugation, 5.28 mLCAC-20K stock solution was slowly mixed into 4.4 mL biphalin stocksolution drop by drop under rapid stirring. 4.4 mL 10×PBS buffer wasadded into the reaction mixture to maintain a relatively neutral pHduring the reaction (measured at 6.8). The CAC-20K to biphalin molarratio was 3.0 with CAC-20K in excess. The reaction was allowed toproceed for 360 min at 21° C. and 12 h at 4° C. for completion. Theformation of (CAC-20K)-2-biphalin was confirmed by analytical RP-HPLC.

TABLE BIP4.1 Analytical RP-HPLC method used to monitor(CAC-20K)₂-biphalin production. Column: Waters Xbridge C18 5 μm 4.6 ×160 mm. Mobile Phase A: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN. Columntemperature: 40° C. UV_(280 nm) was used to follow the elution. TIME(min) % Mobile phase B Flow rate (mL/min) 0.0 20 1.0 5 30 1.0 35 60 1.040 80 1.0 41 20 1.0

The (CAC-20K)₂-biphalin was purified by a CG-71S reverse phase resinusing an AKTA Basic System. The reaction mixture was first diluted 5fold with solvent A [0.1% TFA in water] to reduce sample viscosity. Thediluted sample mixture was loaded onto the CG-71S column at 10 mL/min.After sample loading, the column was first washed with 1 CV solvent A.This was followed by a 30% solvent B [0.1% TFA in acetonitrile] washuntil the UV_(280nm) absorbance remained constant with time. Two peaks,I and II, were eluted during this step. The column was further washedwith 45% solvent B until UV_(280nm) became constant with time. Peak IIIwas eluted in this step. The column was finally washed with 80% solventB. The flow rate was constant at 10.25 ml/min throughout thepurification process. The chromatogram of the loading and elution isshown in FIG. BIP4.1.

The CG-71S column fractions in peaks I, II and III were analyzed by theanalytical RP-HPLC method (Table 1). Unexpectedly, peak I comprisedhighly pure (CAC-20K)₂-biphalin. Most of the free PEG and(CAC-20K)₁-biphalin was washed off in peak II. Although the(CAC-20K)₂-biphalin was the major component in peak III, there was asignificant amount of contamination by free PEG and(CAC-20K)_(I)-biphalin. The average (CAC-20K)-2-biphalin purity in thefractions comprising peak III was estimated at ˜80% by RP-HPLC. Toachieve a higher (CAC-20K)₂-biphalin purity, the peak III fractions werereloaded onto the CG-71S column and a linear gradient elution was usedfor a better separation. The peak III fractions (28 to 31) were pooledand diluted 5 fold with solvent A. The diluted sample mixture was loadedonto the CG-71S column at 10 mL/min. After sample loading, the columnwas first washed with 1 CV solvent A. A gradient elution of 30 to 45%solvent B was next applied within 15 CV. The flow rate was constant at10.25 ml/min throughout the purification process. The chromatogram ofthe loading and elution is shown in FIG. BIP4.2.

The CG-71S column fractions were analyzed by the analytical RP-HPLCmethod (Table). Based on their purities, fractions 23 to 35 were pooled.The purified (CAC-20K)₂-biphalin pool was lyophilized to removeacetonitrile. The lyophilized pellet was reconstituted into 4 mL 20 mMacetate buffer, pH 4.0. The biphalin concentration in the reconstituted(CAC-20K)₂-biphalin was measured to be 0.93 mg/mL by BCA. The purity wasdetermined at 96.8% by RP-HPLC (FIG. BIP4.3). The number-averagemolecular weight was calculated to be 40952.9 Da by MALDI-TOF (FIG.BIP4.4). A final yield of 3.1 mg purified (CAC-20K)₂-biphalin wasobtained.

FIG. BIP4.4: MALDITOF analysis of reconstituted (CAC-20K)₂-biphalin. Thepeak at ˜43 kDa is the expected mass for diPEGylated biphalin. The peakat ˜22 kDa is the expected peak for doubly charged diPEGylated biphalin.The ˜34 KDa MALDI possibly corresponds to diPEGylated biphalin in whichone CAC FMOC group has a single PEG chain.

E BIP5 PEGylation of Biphalin with N-m-PEG-Benzamide-p-SuccinimidylCarbonate (m-PEG-SBC-30K

The conjugation reaction took place in an aqueous environment. 0.84 mgbiphalin was first dissolved into 0.47 mL PBS buffer to make a 1.8 mg/mLbiphalin solution. To initiate the conjugation, 83.2 mg SBC-30K powderwas directly added into 0.47 mL biphalin solution under rapid stirring.The SBC-30K to biphalin molar ratio was 3:0 with SBC-30K in excess. Thereaction was allowed to proceed for 20 min at 21° C. After 20 minutes,0.47 mL 200 mM sodium acetate pH 4.5 buffer was added to stabilize thedi-conjugate. The formation of (SBC-30K)₂-biphalin was confirmed usingan analytical RP-HPLC method (Table 1). The RP-HPLC elution profile isshown in FIG. BIP5.1.

TABLE BIP5.1 Analytical RP-HPLC method used to monitor(SBC-30K)₂-biphalin production Column: Agilent 300Extend-C18 5 μm 4.6 ×250 mm. Mobile Phase A: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN. Columntemperature: 40° C. UV_(280 nm) was used to follow the elution. TIME(min) % Mobile phase B Flow rate (mL/min) 0.0 20 1.0 5 30 1.0 35 60 1.040 80 1.0 41 20 1.0FIG. BIP5.2. The formation of (SBC-30K)₂-biphalin was also confirmed bySDS-PAGE: SDS-PAGE analysis of SBC-30K and biphalin conjugation reactionmixture. The purification of (SBC-30K)₂-biphalin from the reactionmixture was not successful due to the instability of the conjugate, evenat acidic pH values.

E BIP6 Radioligand Binding Assay for Biphalin Series at Delta, Mu, andKappa Opioid Receptors

The binding affinities of biphalin (control) and PEG-biphalin releasableand stable conjugates were evaluated using radioligand binding assays inmembranes prepared from cells expressing recombinant human μ or δ opioidreceptors.

Competition binding experiments were conducted by incubating membraneprotein to equilibrium in triplicate in the presence of a fixedconcentration of radioligand and increasing concentrations (0.1 nM to 10μM) of test compound in 100 μL final volume. The radioligands used werespecific for each receptor type, and the assay conditions are describedin Table BIP6.3. Following incubations, the membranes were rapidlyfiltered through GF/B filter plate (presoaked with 0.5%polyethyleneimine), washed four times with cold 50 mM Tris-HCl, pH 7.5,and the bound radioactivity was then measured. Non-specific binding wasmeasured in the presence of excess naloxone (100 μM); this value wassubtracted from the total binding to yield the specific binding at eachtest concentration.

For all releasable PEG-biphalin conjugates, exceptdi-mPEG-SBC-30K-biphalin, the receptor-binding activity of both releasedbiphalin and PEG-biphalin (unrelased) conjugates were tested. The testcompounds were stored under acidic condition to stabilize the PEGconjugation. To test the activity of PEG-biphalin conjugates, the samplewas diluted on the day of the assay. To test the activity of releasedbiphalin, the sample was diluted 10-fold in assay buffer prior to theassay and pre-incubated under physiological-like conditions for a perioduntil ˜50% of biphalin was estimated to be released, based onpre-determined release rates (refer to T BIP6.4).

IC₅₀ (concentration of test compound required to inhibit 50% of specificbinding) values were obtained from non-linear regression analysis ofdose-response curves, using GraphPad's Prism 5.01 software, and werecalculated for those compounds that showed >50% inhibition of specificbinding at the highest concentration tested. K_(i) (affinity of testcompound) was obtained using the Cheng Prusoff correction usingexperimental K_(d) (affinity of radioligand) values that were previouslydetermined under these assay conditions. The binding affinities ofbiphalin and PEG-biphalin conjugates are shown in Tables BIP6.1 andBIP6.2. Biphalin displayed similar, high affinity (3.1-6.5 nM) for humanμ and δ opioid receptors, and results were comparable to data publishedin literature.

Since the releasable conjugates were pre-incubated in assay buffer, pH7.5 at 37° C., biphalin was also pre-incubated for the maximum time totest the activity of the peptide during treatment underphysiological-like conditions. Biphalin remained stable following 72hour incubation as shown in FIG. 1. Pre-incubated biphalin displayedsimilar, high affinity for μ and δ opioid receptors when compared to thecontrol prepared on the day of the assay (Table BIP6.1).

Following pre-incubation of di-CAC-PEG2-20K-biphalin for 72 hours anddi-C2-PEG2-20K-biphalin for 20 hours, affinity for μ and δ opioidreceptors was increased (compared to PEG-biphalin conjugates prepared onthe day of the assay) and regained (FIG. BIP6.1 and BIP6.2); biphalinreleased from these conjugates retained receptor binding activity asshown by only <4-fold loss in affinity relative to biphalin. Becausedi-mPEG-SBC-30K-biphalin was known to dissociate rapidly, only thesample pre-incubated for 20 hours was tested. Biphalin released from theSBC linker displayed a 16-fold loss in affinity for μ opioid receptorrelative to biphalin; this reduction in affinity may be attributed tothe “tag” contained at the PEG conjugation site of biphalin followingits release. Affinity was not obtained for the δ opioid receptor as >50%inhibition of specific binding was not achieved at the highest testconcentration (1 μM).

The di-CAC-PEG2-20K-biphalin conjugate displayed much lower affinity forboth receptors; reduction in affinity was 324 to 649-folds less relativeto biphalin. The di-C2-PEG2-20K-biphalin conjugate displayed a 5-foldreduction in affinity at the μ opioid receptor and 41-fold reduction atthe δ opioid receptor; this moderate reduction in affinity suggests thatthe di-C2-PEG2-20K linker may have been unstable in the assay buffer andresulted in faster release of biphalin. Furthermore, thedi-C2-PEG2-20K-biphalin conjugate seemed to be more selective for μopioid receptor compared to δ opioid receptor. The receptor selectivitymay have been due to the rate at which each C2-PEG2-20K linker was beingreleased. One hypothesis is that the C2-PEG2-20K conjugated on residue 8was released faster (creating the mono-PEG species conjugated onresidue 1) thereby exposing biphalin's structure to specificallyinteract with the μ opioid receptor site.

As for the stable di-mPEG-SPA-2K conjugate, the loss in affinity for μand δ opioid receptors was significantly greater as shown in FIG.BIP6.2A and BIP6.2B. Binding affinity could not be determined because nomeasurable inhibition of specific binding was detected at the highesttest concentration (10 μM).

FIG. BIP6.1. Competition binding assay of biphalin anddi-CAC-20K-biphalin (released and unreleased) conjugate at human (A) μopioid and (B) δ opioid receptors. Data presented as mean (±SEM) percentspecific binding.FIG. BIP6.2. Competition binding assay of biphalin anddi-C2-20K-biphalin (released and unreleased), di-SBC-30K-biphalin(released), and di-SPA-2K-biphalin (stable) conjugate at human (A) μopioid and (B) δ opioid receptors. Data presented as mean (±SEM) percentspecific binding.

TABLE BIP6.1 Summary of binding affinity for di-CAC-20K-biphalinconjugate. μ Opioid Receptor δ Opioid Receptor Fold Change Fold ChangeRelative to Relative to Test Compound Ki (nM) Biphalin Ki (nM) BiphalinBiphalin 3.4 1.0 6.4 1.0 Biphalin 3.1 0.9 6.5 1.0 (Pre-incubated)Di-CAC-PEG2-FMOC- 1117.0 324.1 4177.0 648.5 NHS-20K-biphalinDi-CAC-PEG2-FMOC- 13.7 4.0 16.8 2.6 NHS-20K-biphalin (Pre-incubated)

TABLE BIP6.2 Summary of binding affinity for di-C2-20K-biphalin,di-SPA-2K-biphalin, and di-SBC-30K-biphalin conjugates. μ OpioidReceptor δ Opioid Receptor Fold Change Fold Change Relative to Relativeto Test Compound Ki (nM) Biphalin Ki (nM) Biphalin Biphalin 4.7 1.0 5.81.0 Di-C2-PEG2- 21.7 4.6 234.9 40.7 FMOC-NHS-20K- biphalin Di-C2-PEG2-10.1 2.1 21.1 3.7 FMOC-NHS- 20K-biphalin (Pre-incubated) Di-mPEG-SBC-77.7 16.4 Not Not obtained 30K-biphalin obtained (Pre-incubated)Di-mPEG-SPA-2K- Not Not obtained Not Not obtained biphalin obtainedobtainedNot obtained=K_(i) values could not be determined since >50% inhibitionof specific binding was not achieved at the highest concentrationtested.

TABLE BIP6.3 Assay conditions. Non- Receptor Membrane specific ReceptorSource Protein Radioligand K_(d) binding Methods μ Opioid Human  5μg/well [³H] 4.0 nM Naloxone Reaction in 50 mM Tris- recombinantNaloxone (100 μM) HCl (pH 7.5) at 25° C. for CHO-K1 (5 nM) 1 h on plateshaker cells δ Opioid Human 15 μg/well [³H] 3.9 nM Naloxone Reaction in50 mM Tris- recombinant DPDPE (100 μM) HCl (pH 7.5), 5 mM CHO-K1 (5 nM)MgCl₂, 0.1% BSA at cells 25° C. for 1 h on plate shaker

TABLE BIP6.4 Test compounds. Stock concentration PEG ReleasePre-incubation Test based on peptide Storage rate (if condition (ifCompound MW (Da) (mg/mL) buffer applicable) applicable) Biphalin 909 5.0PBS, pH — 72 h in 50 mM 7.4 Tris-HCl, 5 mM MgCl2, 0.1% BSA, pH 7.5 at37° C. Di-CAC- 40,952 0.93 20 mM 15% after 23 h 72 h in 50 mM PEG2-FMOC-acetate, at 37° C. in Tris-HCl, 5 mM NHS-20K- pH 4.0 PBS, pH 7.2 MgCl2,biphalin 0.1% BSA, pH Releasable 7.5 at 37° C. Di-C2-PEG2- 42,055 0.9920 mM 50% after 4 h 20 h in 50 mM FMOC-NHS- acetate, at 37° C. inTris-HCl, 5 mM 20K-biphalin pH 4.0 PBS, pH 7.2 MgCl2, Releasable 0.1%BSA, pH 7.5 at 37° C. Di-mPEG- 63,920 2.7 20 mM 55% after 50 min 20 h in50 mM SBC-30K- acetate, at 37° C. in Tris-HCl, 5 mM biphalin pH 4.0 200mM Na- MgCl2, Releasable phosphate, pH 0.1% BSA, pH 7.4 7.5 at 37° C.Di-mPEG- 5,394 7.92 20 mM — — SPA-2K- acetate, biphalin pH 4.0 Stable

E BNP1 BNP-mPEG Conjugates

a) mPEG-N^(ter)-BNP Via mPEG-SPC

BNP is prepared and purified according to standard automated peptidesynthesis or recombinant techniques known to those skilled in the art.An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of BNP, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of BNP prepared in phosphate buffered saline, PBS, pH 7.4 isadded and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-BNPconjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) BNP-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of BNP, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected BNP(Prot-BNP) is prepared and purified according to standard automatedpeptide synthesis techniques known to those skilled in the art. mPEG-NH₂20 kDa, stored at −20° C. under argon, is warmed to ambient temperature.The reaction is performed at room temperature. About 5-fold molar excessof mPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-BNP is prepared in N,N-dimethylformamide is added and the mixtureis stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-BNP-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theBNP-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) BNP-Cys(S-mPEG)

mPEG-Maleimide is obtained having a molecular weight of 5 kDa and havingthe basic structure shown below:

mPEG-MAL, 5 kDa

BNP, which has a thiol-containing cysteine residue, is dissolved inbuffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-BNP Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock BNP solution and mixed well. After the addition of the mPEG-SMB,the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8using conventional techniques. To allow for coupling of the mPEG-SMB tothe peptide via an amide linkage, the reaction solution is stirred forseveral hours (e.g., 5 hours) at room temperature in the dark or stirredovernight at 3-8° C. in a cold room, thereby resulting in a conjugatesolution. The reaction is quenched with a 20-fold molar excess (withrespect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) BNP-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of BNP, to provide a Glu-conjugate form ofthe peptide. For coupling to the Glu residue, a protected BNP (Prot-BNP)is prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Glu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate forsubsequent coupling. mPEG-NH₂ 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. A 5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-BNP is prepared inN,N-dimethylformamide is added and the mixture is stirred using amagnetic stirrer until the mPEG-NH₂ is fully dissolved. The stirringspeed is reduced and the reaction is allowed to proceed to formation ofconjugate product. The conjugate solution is then analyzed by SDS-PAGEand RP-HPLC (C18) to determine the extent of Prot-BNP-(Glu-O-mPEG)conjugate formation. The remaining protecting groups are removed understandard deprotection conditions to yield the BNP-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

E BNP2 PEGylation of BNP-32 with mPEG2-Butyr-ALD-40K

A BNP-32 stock solution of 4 mg/mL peptide content was made in 20 mMNa-citrate buffer pH 4.5 in a sterile low-endotoxin polypropylene tube.This solution could be stored aseptically for at least 1 week at 4° C.Immediately before a PEGylation reaction was performed, a 100 mg/mLstock solution of mPEG-Butyr-ALD-40K was made in the same buffer. A 50mg/mL solution of sodium-cyanoborohydride (Na—CNHBr) reducing reagent inMilli-Q water was also made immediately before use. A typical PEGylationreaction was carried out as follows: Peptide stock solution (3 mL) wastransferred to an appropriate tube containing a magnetic stir-bar and5.208 mL of the same buffer was added. While stirring, 3.672 mL of a 100mg/mL solution of mPEG-Butyr-ALD 40K was added dropwise within 1 minute.The reaction was allowed to stir for 15 min after which 0.12 mL of a 50mg/mL Na—CNHBr solution was added, and the reaction mixture allowed tostir overnight (16-18 h) at room temperature. The resultant reactionmixture contained 1 mg/mL peptide, 2.0 mol equivalents of PEG (withrespect to peptide) and 10 mol equivalents of NaCNBr (with respect toPEG). The reaction rate analysis is shown in FIG. BNP2.1. The reactionyields were determined by reversed phase HPLC to be 80.4% mono-PEGconjugate (N-terminus directed), 8.9% di-PEG conjugate and 10.7%non-conjugated peptide.

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using a Hi Trap SP Sepharose HP media (GEHealthcare). The linear flow rate of the column was 150 cm/h and thesample loading was 2.0 mg/mL of column bed volume (CV) with a column bedheight of 10 cm. The buffers used for purification were: Buffer A: 10 mMNaPO₄, pH 7.0 and Buffer B: Buffer A+0.5 M NaCl.

The PEGylation reaction mixture was diluted with 4 volumes of buffer Aand the pH adjusted to 8.0. The column was equilibrated in buffer A. Thediluted reaction mixture was loaded onto the column and unboundsubstances washed off the column with 3 column volumes of buffer A. Theconjugated peptide was eluted from the column using a linear gradient of0-100% B over 10 CV. A typical chromatogram is shown in FIG. BNP2.2. Thepurity of the conjugate was 99.5% (by RP-HPLC analysis, FIG. BNP2.3) andthe mass (as determined by MALDI-TOF, FIG. BNP2.4) was within theexpected range. The detection wavelength for preparative and analyticalchromatography was 225 nm.

Samples were analyzed using reversed-phase HPLC. The mobile phases wereA, 0.1% TFA in water and B, 0.05% TFA in acetonitrile. An AgilentPoroshell 300-SB-C8 (P/N 660750-906) column was used with a flow of 0.5ml/min and column temperature of 50° C. The column was equilibrated in10% B and conjugate separation was achieved using the gradient timetableshown in T BNP2.1 below.

Time (min) % B 0  10 2  10  5.5 45 10.5 65 10.6 95 13.6 95 13.7 10 Postrun 5 min

FIG. BNP2.1. PEGylation rate of BNP-32 with mPEG2-40kDa Butyr-ALD. Thereaction yields were 80.4% mono-PEG conjugate, 8.9% di-PEG conjugate and10.7% remaining non-PEGylated peptide after 18 h reaction time. Yieldswere determined by RP-HPLC.

FIG. BNP2.2. Typical purification profile for the 40 kDa mPEG2-Butyr-ALDmono-PEG conjugate of BNP-32. The mono-PEGylated conjugate is indicated.The di-PEG conjugate eluted during the loading step.

FIG. BNP2.3. HPLC analysis of the 40 kDa mPEG2-Butyr-ALD mono-PEGconjugate of BNP-32. The mono- and di-PEGylated forms of BNP-32 areindicated. The peak at 8 min retention time was instrument related andnot any product of interest.

FIG. BNP2.4. MALDI-TOF analysis of the 40 kDa mPEG2-Butyr-ALD mono-PEGconjugate of BNP-32. The detected mass of the major peak was 45138 Da,which was within the expected range for the mono-conjugate.

FIG. BNP2.5. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofBNP-32 and purified [mono]-[mPEG2-Butyr-ALD-40K]-[BNP-32] conjugate.Lanes 1, 2 and 3 are 0.5, 1.0 and 2.0 μg of the non-PEGylated peptiderespectively. Lanes 4, 5 and 6 are 0.5, 1.0 and 2.0 μg of the purifiedmono-PEG-conjugate, respectively.

E BNP3 Site Specific Acetylation of Brain Natriuretic Peptide (BNP-32)

Specific amine sites can be blocked by acetylation leaving other sitesopen for PEGylation. BNP-32 is composed of 32 amino acids with a singledisulfide bond. The peptide contains 3 lysine residues and an N-terminuscontaining a free amine group. Previous PEGylation studies with BNP-32indicate that all four amine groups are sterically accessible forreaction with PEG reagents. (Miller et al., Bioconjugate Chemistry 2006March-April; 17(2):267-74). In the current study, the pKa differencebetween the N-terminal amine and the epsilon amines of the lysineresidues was used to specifically acetylate the N-terminus, leaving thelysine amines available for PEGylation.

One milligram of BNP-32 was combined with 2 mol equivalents of aceticacid-NHS (previously dissolved in 2 mM HCl) in a total volume of 1 mL in20 mM MES buffer at pH 6.0 and incubated at room temperature for 2 h. Atthis pH, one predominant acetylated product was formed based on RP-HPLCanalysis. Based on accepted chemical principles known to those skilledin the art, at pH 6.0 the N-terminal amine group is more reactive thanthe epsilon amines and acetylation would occur predominantly at thisposition. Also, at lower pH, all amines are less reactive while athigher pH all amines are more reactive. The reaction above was alsoperformed at other pH levels: At pH 4.5 (20 mM citrate buffer) there wassignificantly lower acetylation for all amine groups, while at pH 7.5(20 mM HEPES buffer) and pH 9.0 (20 mM boric acid buffer), all aminegroups were more reactive and significant acetylation occurred at allfour sites as assed by RP-HPLC. Site specificity of the purifiedreaction products may also be confirmed using methods known to the artsuch as peptide mapping.

The predominant acetylated product from the reaction performed at pH 6.0can be purified by standard chromatographic methods. The acetylatedpeptide can then be PEGylated using any of the reagents that arespecific for amine reactive groups and standard methods known to theart, again followed by standard chromatographic methods to purify theconjugate of interest.

E BNP4 PEGylation of BNP-32 with [mPEG-Butyr-ALD-10K]

A BNP-32 stock solution of 4 mg/mL peptide content was made in 20 mMsodium-citrate buffer pH 4.5 in a sterile low-endotoxin polypropylenetube. This solution could be stored aseptically for at least 1 week at4° C. Immediately before a PEGylation reaction was performed, a 100mg/mL stock solution of [mPEG-Butyr-ALD-10K] was made in the same bufferused to dissolve the peptide. A 50 mg/mL solution ofsodium-cyanoborohydride (Na—CNHBr) reducing reagent in Milli-Q water wasalso made immediately before use. A typical PEGylation reaction wascarried out as follows: Peptide stock solution (3 mL, 12 mg) wastransferred to an appropriate tube containing a magnetic stir-bar and8.11 mL of 20 mM sodium-citrate buffer pH 4.5 was added. While stirring,0.77 mL of a 100 mg/mL solution of mPEG-Butyr-ALD 10K was added dropwise within 1 minute. The reaction was allowed to stir for 15 min afterwhich 0.12 mL of a 50 mg/mL Na—CNHBr solution was added, and thereaction mixture allowed to stir overnight (16-18 h) at roomtemperature. The resultant reaction mixture contained 1 mg/mL peptide,2.0 mol equivalents of PEG (with respect to peptide) and 10 molequivalents of NaCNBr (with respect to PEG). The reaction yields weredetermined by reversed phase HPLC to be 76% mono-PEG conjugate(N-terminus directed), 10.6% di- and tri-PEG conjugate and 13.4%non-conjugated peptide. This PEG reagent forms stable bonds with aminegroups.

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using Hi Trap SP Sepharose HP media (GEHealthcare). The linear flow rate of the column was 150 cm/h and thesample loading was 2.0 mg/mL of column bed volume (CV) with a column bedheight of 10 cm. The buffers used for purification were: Buffer A: 10 mMNaPO₄, pH 7.0 and Buffer B: Buffer A+0.5 M NaCl. The PEGylation reactionmixture was diluted with 4 volumes of buffer A and the pH adjusted to8.0 with 0.1 M sodium hydroxide. The column was equilibrated in bufferA. The diluted reaction mixture was loaded onto the column and unboundsubstances washed off the column with 3 column volumes of buffer A. Theconjugated peptide was eluted from the column using a linear gradient of0-100% B over 10 CV. The detection wavelength for preparative andanalytical chromatography was 225 nm.

Fractions collected during cation exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were: A, 0.1% TFA in waterand B, 0.05% TFA in acetonitrile. An Agilent Poroshell 300-SB-C8 (P/N660750-906) column was used with a flow of 0.5 ml/min and columntemperature of 50° C. The column was equilibrated in 10% B and conjugateseparation was achieved using the gradient timetable shown in Table 2.1.

Time (min) % B 0  10 2  10  5.5 45 10.5 65 10.6 95 13.6 95 13.7 10 Postrun 5 min

Fractions containing pure [mono]-[mPEG-Butyr-ALD-10K]-[BNP-32] asdetermined by RP-HPLC were pooled and stored in aliquots at −80° C. asthe purified conjugate.

A typical cation-exchange chromatogram is shown in FIG. BNP4.1. SDS-PAGEanalysis of BNP-32 and purified [mono]-[mPEG2-Butyr-ALD-10K]-[BNP-32]conjugate is shown in FIG. BNP4.2. RP-HPLC analysis of the purifiedconjugate is shown in FIG. BNP4.3, and MALDI-TOF analysis of thepurified conjugate is shown in FIG. BNP4.4. The purity of themono-PEG-conjugate was 98% by SDS-PAGE analysis and 98.4% by RP-HPLCanalysis with 1.6% of di-PEG-conjugate. The mass as determined byMALDI-TOF was within the expected range.

FIG. BNP4.1. Typical cation-exchange purification profile of[mono]-[mPEG-Butyr-ALD-10K]-[BNP-32]. The PEGylated conjugates and thefree peptide peaks are indicated.

FIG. BNP4.2. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofBNP-32 and the purified [mono]-[mPEG2-Butyr-ALD-40K]-[BNP-32] conjugate.Lane 1: BNP-32 peptide only (1 μg); Lanes 2, 3 and 4 are 0.5, 1.0 and2.0 μg of the purified mono-PEG-conjugate, respectively.

FIG. BNP4.3. RP-HPLC analysis of the purified[mono]-[mPEG-Butyr-ALD-10K]-[BNP-32] conjugate. The peaks at 7.851 and8.396 min contain the mono-PEG and di-PEG conjugates, respectively.

FIG. BNP4.4. MALDI-TOF analysis of the purified[mono]-[mPEG-Butyr-ALD-10K]-[BNP-32] conjugate. The detected mass of themajor peak was 14568 Da, which was within the expected range for themono-PEG conjugate. The peak at 7232 Da represents the doubly chargedconjugate.

E BNP5 PEGylation of BNP-32 with releasable [mPEG-SBC-30K]

A BNP-32 stock solution of 4 mg/mL peptide content was made in 20 mM MESbuffer pH 6.0 in a sterile low-endotoxin polypropylene tube. Thissolution could be stored aseptically for at least 1 week at 4° C.

A typical PEGylation reaction was carried out as follows: [mPEG-SBC-30K]PEG reagent (1220 mg) was weighed-out in an appropriate tube anddissolved with stirring in 9 ml of the same buffer used to dissolve thepeptide. After the PEG had dissolved and with stirring, 3.0 mL of thepeptide solution was added. The reaction was allowed to stir for 10 minat room temperature. The resultant reaction mixture contained 1 mg/mLpeptide and 8.0 mol equivalents of PEG. After the incubation period, 1/9volume of a 1 M glycine solution (in the same buffer) was added toquench the reaction. After a further 60 min of stirring at roomtemperature, 1 volume of 0.2 M acetic acid was added to stabilize theconjugate and the reaction mixture was stored at −20° C. The reactionyielded >80% mono-PEG conjugate. The mPEG SBC reagent forms hydrolysablebonds with amine groups and upon hydrolysis, leaves the peptide modified(tagged).

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using Hi Trap SP Sepharose HP media (GEHealthcare). The linear flow rate of the column was 150 cm/h and thesample loading was 2.0 mg/mL of column bed volume (CV) with a column bedheight of 10 cm. The buffers used for purification were: Buffer A: 10 mMNaPO₄, pH 7.0 and Buffer B: Buffer A+0.5 M NaCl. The PEGylation reactionmixture was diluted with 4 volumes of buffer A and the pH adjusted to8.0 with 0.1 M sodium hydroxide. The column was equilibrated in bufferA. The diluted reaction mixture was loaded onto the column and unboundsubstances washed off the column with 3 column volumes of buffer A. Theconjugated peptide was eluted from the column using a linear gradient of0-100% B over 10 CV. The pooled mono-PEGylated fraction was diluted with4 volumes of buffer A and the purification step repeated. The detectionwavelength for preparative and analytical chromatography was 225 nm.

Fractions collected during cation exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were A, 0.1% TFA in waterand B, 0.05% TFA in acetonitrile. An Agilent Zorbax 5 μm 300-SB-C18,4.5×50 mm (P/N 860950-902) column was used with a flow of 1.0 ml/min andcolumn temperature of 60° C. The column was equilibrated in 10% B andconjugate separation was achieved using the gradient timetable shown inT BNP5.1 below.

Time (min) % B 0  10 2  10 4  30 8  34 10.2 56 16.2 62 16.3 90 17.0 90 17.01 10 Post run 5 min

Fractions containing pure [mono]-[mPEG-SBC-30K]-[BNP-32] from the repeatcation-exchange chromatography as determined by RP-HPLC were pooled andstored in aliquots at −80° C. as the purified conjugate.

A typical cation-exchange purification chromatogram is shown in FIG.BNP5.1. SDS-PAGE analysis of purified [mono]-[mPEG-SBC-30K]-[BNP-32] isshown in FIG. BNP5.2. RP-HPLC analysis of the purified conjugate isshown in FIG. BNP5.3, and MALDI-TOF analysis of the purified product isshown in FIG. BNP5.4. The purity of the mono-PEG-conjugate was 95.8% byRP-HPLC analysis with 4.2% di-PEG conjugate also present. The mass asdetermined by MALDI-TOF was within the expected range.

FIG. BNP5.1. Typical first cation-exchange purification profile for[mono]-[mPEG-SBC-30K]-[BNP-32]. The mono- and di-PEGylated conjugatesare indicated. The free peptide eluted in two peaks. On release, thisPEG reagent leaves a modified (tagged) peptide. Peak 1 and peak 2contain modified and unmodified peptide, respectively.

FIG. BNP5.2. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofthe purified [mono]-[mPEG-SBC-30K]-[BNP-32] conjugate. Lanes 1, 2 and 3are 0.7, 1.4 and 2.1 μg of the PEGylated peptide, respectively.

FIG. BNP5.3. RP-HPLC analysis of the purified[mono]-[mPEG-SBC-30K]-[BNP-32] conjugate. The peaks at 12.041 min and12.726 retention times contain the mono-PEG and di-PEG conjugates,respectively.

FIG. BNP5.4. MALDI-TOF analysis of the purified[mono]-[mPEG-SBC-30K]-[BNP-32] conjugate. The detected mass of the majorpeak was 32580 Da, which was within the expected range for themono-PEG-conjugate. The peak at 17444 Da represents the doubly chargedconjugate.

E BNP6 PEGylation of BNP-32 with [mPEG2-C2-fmoc-NHS-40K]

A BNP-32 stock solution of 4 mg/mL peptide content was made in 20 mM MESbuffer pH 5.8 in a sterile low-endotoxin polypropylene tube. Thissolution could be stored aseptically for at least 1 week at 4° C.

Immediately before a PEGylation reaction was performed, a 100 mg/mLstock solution of [mPEG2-C2-fmoc-NHS-40K] PEG reagent was made in thesame buffer used to dissolve the peptide. A typical PEGylation reactionwas carried out as follows: Peptide stock solution (6 mL, 24 mg) wastransferred to an appropriate tube containing a magnetic stir-bar and10.16 mL of 20 mM MES buffer pH 5.8 was added. While stirring, 7.84 mLof a 100 mg/mL PEG reagent solution was added. The resultant reactionmixture contained 1 mg/mL peptide and 2 mol equivalents of PEG. Thereaction was allowed to stir for 90 min at room temperature after whicha 1/9 volume of 0.2 M glycine solution (in 20 mM MES buffer pH 5.8) wasadded and the reaction mixture stirred for another 60 min to quench thereaction. These reaction conditions yielded approximately 60%mono-PEGylated peptide. This PEG reagent forms hydrolysable bonds withamine groups and upon hydrolysis, an unmodified peptide is generated.The reaction mixture was stored at 4° C.

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using Hi Trap SP Sepharose HP media (GEHealthcare). The linear flow rate of the column was 150 cm/h and thesample loading was 1.0 mg/mL of column bed volume (CV) with a column bedheight of 11 cm. The buffers used for purification were: Buffer A: 10sodium-citrate, pH 4.0 and Buffer B: Buffer A+0.8 M NaCl. The PEGylationreaction mixture was diluted with 4 volumes of buffer A. The column wasequilibrated in buffer A. The diluted reaction mixture was loaded ontothe column and unbound substances washed off the column with 3 columnvolumes of buffer A. The conjugated peptides were eluted from the columnusing the following elution steps: (a) linear gradient of 0-4% B over 1CV followed by a hold at 4% B for 4 CV; (b) linear gradient of 4-50% Bover 5 CV followed by a hold at 50% B for 1 CV; (c) step gradient to 80%B followed by a hold at 80% B for 2 CV. The pooled mono-PEGylatedfraction was diluted with 4 volumes of buffer A and the purificationstep repeated. The detection wavelength for preparative and analyticalchromatography was 225 nm.

Fractions collected during cation exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were A, 0.1% TFA in waterand B, 0.05% TFA in acetonitrile. An Agilent Zorbax XDB-C8, 5 μm,4.5×150 mm (P/N 993967-906) column was used with a flow of 0.5 ml/minand column temperature of 60° C. The column was equilibrated in 10% Band conjugate separation was achieved using the gradient timetable shownin T BNP6.1 below.

Time (min) % B  0 10  4 10  9 35   10.5 50 23 75 24 95 25 95   25.2 10Post run 6 min

Fractions containing pure [mono]-[mPEG2-C2-fmoc-NHS-40K]-[BNP-32] fromthe repeat cation-exchange chromatography as determined by RP-HPLC werepooled and stored in aliquots at −80° C. as the purified conjugate.

A typical first cation-exchange purification chromatogram is shown inFIG. BNP6.1. SDS-PAGE analysis of purified[mono]-[mPEG2-C2-fmoc-NHS-40K]-[BNP-32] is shown in FIG. BNP6.2. RP-HPLCanalysis of the purified conjugate is shown in FIG. BNP6.3, andMALDI-TOF analysis of the purified conjugate is shown in FIG. BNP6.4.The purity of the mono-PEG-conjugate was 100% by RP-HPLC analysisand >95% (by SDS-PAGE). The mass as determined by MALDI-TOF was withinthe expected range.

FIG. BNP6.1. Typical first cation-exchange purification profile of[mPEG2-C2-fmoc-NHS-40K]. The mono-, di- and non-PEGylated (free peptide)elution peaks are indicated.

FIG. BNP6.2. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofthe purified [mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate. Lanes 1 and 2are 1.0 and 2.0 pg of the PEGylated peptide, respectively. Low levels ofhi-PEGylated forms are also visible.

FIG. BNP6.3. RP-HPLC analysis of the purified[mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate. FIG. BNP6.4. MALDI-TOFanalysis of the purified [mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate.

FIG. BNP6.4. MALDI-TOF analysis of the purified[mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate. The detected mass of themajor peak was 44725 Da, which was within the expected range for themono-PEG-conjugate.

E BNP7 Pharmacokinetic Studies

Thirty one (31) adult male Sprague-Dawley rats with indwelling jugularvein and carotid artery catheters (JVC/CAC) (Charles River Labs,Hollister, Calif.) were utilized for this study. The weight range of theanimals was 315-358 grams. All animals were food fasted overnight. Priorto dosing, the rats were weighed, the tails and cage cards were labeledfor identification and the doses were calculated. Anesthesia was inducedand maintained with 3.0-5.0% isoflurane. The JVC and CAC wereexternalized and flushed with HEP/saline (10 IU/mL HEP/mL saline). Thepredose sample was collected from the JVC and the catheters wereplugged, and labeled to identify the jugular vein and carotid artery.When all of the animals had recovered from anesthesia and the predosesamples were processed, the animals were dosed, intravenously (IV) viathe JVC using a 1 mL syringe containing the appropriate test article andthe dead volume of the catheter was flushed with 0.9% saline to ensurethe animals received the correct dose.

Following a single IV dose, blood samples were collected from groups 1A,2A, 3A and 4A, at 0 (pre-dose collected as described above), 0.03, 0.33,2.0, 6.0, 12.0 and 72.0 hours and from Groups 1B, 2B, 3B and 4B at 0(pre-dose collected as described above), 0.17, 1.0, 4.0, 8.0, 24.0 and48.0 hours via the carotid artery catheter and processed as stated inthe protocol. Following the last collection point, the animals wereeuthanized.

Pharmacokinetic Analyses: Noncompartmental PK data analysis and reportpreparation was completed by Research Biology at Nektar Therapeutics(India) Pvt. Ltd. Hyderabad, A.P., India. Individual plasmaconcentration data are listed and summarized in Appendix A1.1-1.3. PKanalysis was performed using WinNonlin (Version 5.2, Mountain View,Calif.-94014). Concentrations in plasma that were below LLOQ werereplaced with zeros prior to generating Tables and PK analysis. In theevent that more than half (>50%) of the data points were below zero,mean concentration will not be shown in the figures or used in PKparameters estimation. The following PK parameters were estimated usingplasma concentration-time profile of each animal:

C0 Extrapolated concentration to time “zero”C_(max) Maximum (peak) concentrationAUCall Area under the concentration-time from zero to time of lastconcentration valueT½(Z) Terminal elimination half-lifeAUCinf Area under the concentration-time from zero to time infinityTmax Time to reach maximum or peak concentration followingadministrationCL Total body clearanceVz Volume of distribution based on terminal phaseVss Volume of distribution at steady stateMRTlast Mean residence time to last observable concentration

Releasable-PEG:

FIG. BNP7.1 shows the mean plasma concentration-time profiles of forC2-FMOC-PEG2-40K-BNP, its corresponding metabolite and released BNP. Nomeasurable plasma concentrations observed after BNP administration andhence the data is not shown in FIG. BNP7.1. At first time pointcollection which was at 0.03 hr, concentration was <20 ng/mL in all theanimals.

Table BNP7.1 summarizes the PK parameters of BNP following equivalentprotein mass of 0.459 mg/kg administered intravenously into rats viaC2-FMOC-PEG2-40K-BNP or BNP.

TABLE 1 Comparative PK Parameters of BNP Released from C2-FMOC-PEG2-40K-BNP in BNP Given as Non-Conjugated Native Protein Cmax T½ AUCINFTmax MRTlast Test Article (ng/mL) (hr) (ng · hr/mL) (hr) (hr) BNP 0.00NC NC NC NC C2-FMOC-PEG2- 55.4 1.25 162 0.33 1.84 40K-BNP NC - Cannot becalculated.FIG. BNP7.2 shows the non-released PEG-BNP levels after theadministration of the two non-releasable PEG constructs(ButyrALD-40K-BNP, ButyrALD-10K-BNP). T BNP7.2 summarizes the PKparameters of following equivalent protein mass of 0.459 mg/kgadministered intravenously into rats.

TABLE BNP7.2 Comparative PK Parameters of Test Articles(Non-Releasable-PEG Conjugates) versus Native BNP Following EquivalentProtein Mass Intravenous Administration to Sprague Dawley rats (Mean ±SD) CL Vss Test Cmax T½ AUCINF MRTlast (mL/ (mL/ Compound (ng/mL) (hr)(ng · hr/mL) (hr) hr/kg) kg) BNP 0.00 NC NC NC NC NC ButyrALD- 1410 26.141300 24.0 11.1 631 40K-BNP ButyrALD- 355 0.272 96.6 0.368 4750 227010K-BNP NC - Cannot be calculated, there were no measurable plasmaconcentrations.

BNP concentrations were <LLOQ (LLOQ: 20 ng/mL) and therefore, no PKParameters were reported.

BNP released from C2-FMOC-PEG2-40K-BNP reached peak concentrations of55.4 ng/mL at 0.3 h and stayed above 20 ng/mL for 8 hr followingC2-FMOC-PEG2-40K-BNP dosing. Half-life value for released BNP is 1.25 hfollowing C2-FMOC-PEG2-40K-BNP IV bolus administration. Peakconcentrations of 1300 ng/mL, a half-life of 15.0 hr and with plasmaC2-FMOC-PEG2-40K-BNP concentrations remained above 100 ng/mL up to 24 hsupported the prolonged release of BNP in plasma. The observed releaseof BNP from releasable-PEG C2-FMOC-PEG2-40K-BNP is consistent with theappearance of free PEG-metabolite (PEG-fulvene) which was also releasedfrom the conjugate. Binding to cell surface clearance receptors withinternalization and degradation, proteolytic cleavage and renalfiltration are the possible route of elimination for releasableC2-FMOC-PEG2-40K-BNP.

For the non-releasable PEG-constructs, ButyrALD-40K-BNP was observed tohave longer half-life, lower clearance and higher exposure thanButyrALD-10K-BNP, probably due to increased PEG-length of the conjugate.No BNP was measurable in plasma following parent BNP administration.

Due to staggered sample collection, two very distinct concentration-timeprofiles were observed for two subgroups received ButyrALD-40K-BNPtreatment. Therefore, the PK parameters estimated from the pooled datafrom the two subgroups to be interpreted with caution. ButyrALD-40K-BNPshowed higher peak plasma concentration, approximately higher exposureand longer half-life than ButyrALD-10K-BNP when compared using pooleddata.

E PRO1 Protegrin-mPEG Conjugates

a) mPEG-N^(ter)-Protegrin Via mPEG-SPC

Protegrin is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of protegrin, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. An X-fold molar excess of mPEG-SPC 20 kDareagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of Protegrin prepared in phosphate buffered saline, PBS, pH7.4 is added and the mixture is stirred using a magnetic stirrer untilthe mPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1 M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent ofmPEG-N^(ter)-Protegrin conjugate formation.Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) Protegrin-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of protegrin, to provide a C^(ter)-conjugateform of the peptide. For coupling to the C-terminus, a protectedProtegrin (Prot-protegrin) is prepared and purified according tostandard automated peptide synthesis techniques known to those skilledin the art. mPEG-NH₂ 20 kDa, stored at −20° C. under argon, is warmed toambient temperature. The reaction is performed at room temperature. AX-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot-protegrin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SD S-PAGE and RP-HPLC (C18) to determine the extent ofProt-Protegrin-C^(ter)-mPEG conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the protegrin-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) Protegrin-Cys(S-mPEG)

mPEG-Maleimide is obtained having a molecular weight of 5 kDa and havingthe basic structure shown below:

mPEG-MAL, 5 kDa

Protegrin, which has a thiol-containing cysteine residue, is dissolvedin buffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-Protegrin Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock protegrin solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Tris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) Protegrin-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of Protegrin, to provide a Glu-conjugateform of the peptide. For coupling to the Glu residue, a protectedProtegrin (Prot2-Protegrin) is prepared and purified according tostandard automated peptide synthesis techniques known to those skilledin the art. Deprotection of the Glu(OBz) residue (H₂/Pd) yields thefree-Glu carboxylate for subsequent coupling. mPEG-NH₂ 20 kDa, stored at−20° C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. A 5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-Protegrin isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-Protegrin-(Glu-O-mPEG) conjugate formation. The remainingprotecting groups are removed under standard deprotection conditions toyield the Protegrin-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

E PRO2 PEGylation of Protegrin-1 (PG-1) with [mPEG2-CAC-FMOC-NHS-40K]

Stock solutions of 5.0 mg/mL PG-1 and 200 mG/mL mPEG2-CAC-FMOC-NHS-40Kwere prepared in 2 mM HCl. To initiate a reaction, the two stocksolutions and a 0.5 M MES, pH 6.0, stock solution were brought to 25° C.and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.0 mg/mLPG-1, 50 mM MES and a 5-fold molarexcess of mPEG2-CAC-FMOC-NHS-40K over PG-1. After 3.5 hours at 25° C.the reaction was quenched with 100 mM glycine in 100 mM HCl (10 mM finalglycine concentration) for 1 hour. The reaction mixture was then dilutedwith deionized sterile water until the conductivity was below 1.0 mS/cmand the pH was adjusted to 6.0 with 1 M Na₂CO₃/NaHCO₃, pH 10.0.

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using a column packed with SPHP media (GEHealthcare) on an AKTA Explorer 100 system (GE Healthcare). Buffer A was20 mM MES, pH 6.0; Buffer B was 20 mM MES and 1 M NaCl, pH 6.0. The AKTAExplorer plumbing system and SPHP column were sanitized with 1 M HCl and1 M NaOH and the resin was equilibrated with 10 column volumes Buffer Aprior to sample loading. After loading, the column was washed with 10column volumes 80% A/20% B to remove un-reacted PEG reagent. PEGylatedand nonPEGylated peptides were eluted using a linear gradient from 80%A/20% B to 0% A/100% B over 20 column volumes with a linear flow rate of90 cm/hour.

Fractions collected during cation exchange chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.1%TFA in water, and B, 0.05% TFA in acetonitrile. A Waters Symmetry C18column (4.6 mm×75 mm) was used with a flow rate of 1.0 ml/min and acolumn temperature of 50° C. Detection was carried out at 280 nm. Thecolumn was equilibrated in 20% B and conjugate separation was achievedusing the gradient timetable shown in T PRO2.1.

Step Time (min) % Mobile phase B 1 0.00 20.0 2 2.00 30.0 3 5.00 45.0 46.00 45.0 5 18.00 80.0 6 18.10 100.0 7 20.10 100.0 8 20.20 10.0

Fractions containing pure mono-[mPEG2-CAC-FMOC-40K]-[PG-1] as determinedby RP-HPLC were pooled. Glacial acetic acid was added to the pooledfractions to a final concentration of 5% (v/v) and loaded onto a CG71Scolumn (Rohm Haas) for Endotoxin removal and buffer exchange. Prior tosample loading, the column had been washed with 5% acetic acid inacetonitrile and equilibrated with 5% acetic acid in water (v/v). Aftersample loading, the column was washed with 10 column volumes of 5%acetic acid and mono-[mPEG2-CAC-FMOC-40K]-[PG-1] was eluted with alinear 0-100% gradient from 5% acetic acid to 5% acetic acid/95%Acetonitrile (v/v) over 10 column volumes. Fractions containing theconjugate as determined by analytical reversed phase HPLC, were pooled,lyophilized and stored at −80° C.

A typical SPHP cation exchange chromatogram is shown in FIG. PRO2.1.SDS-PAGE analysis of purified mono-[mPEG2-CAC-FMOC-40K]-[PG-1] is shownin FIG. PRO2.2. RP-HPLC analysis of the purified conjugate is shown inFIG. PRO2.3, and MALDI-TOF analysis of the purified conjugate is shownin FIG. PRO2.4.

The purity of the mono-PEG-conjugate was >95% by SDS-PAGE and 100% byRP-HPLC analysis. The mass as determined by MALDI-TOF was within theexpected range.

FIG. PRO2.1. Typical cation exchange purification profile ofmono-[mPEG2-CAC-FMOC-40K]-[PG-1]. The mono-PEGylated conjugate,unreacted peptide and PEG are indicated. The blue line representsabsorbance at 280 nm and the red line represents absorbance at 225 nm.

FIG. PRO2.2. SDS-PAGE, with Coomassie Blue staining) of purified[mono]-[CAC-PEG2-FOMC-NHS-40K]-[Protegrin-1]. Lane 1, Mark12 MW markers;Lane 2, purified [mono]-[CAC-PEG2-FOMC-NHS-40K]-[Protegrin-1]. Lane 3,smaller quantity of purified[mono]-[CAC-PEG2-FOMC-NHS-40K]-[Protegrin-1]. The apparent largemolecular weight of the conjugate, about 95 kDa, is due to a slowmobility of the monomeric conjugate in the gel due to a high degree ofPEG hydration. Impurities were not detected in Lane 2.

FIG. PRO2.3. Purity analysis of [mono]-[CAC-PEG2-FOMC-40K]-[Protegrin-1]by Reversed Phase HPLC. The purity of the purified conjugate is 100% %at 280 nm. The peak at 4.5 minutes is a column-derived species and isnot included in the sample.

FIG. PRO2.4. MALDI-TOF spectrum of purifiedmono-[CAC-PEG2-FMOC-40K]-[Protegrin-1]. The major peak at 43.1 kDa iswithin the expected range for the molecular weight of themono-PEG-conjugate. The peak at 85.4 kDa may represent the singlecharged conjugate dimer formed during MALDI-TOF analysis.

E PRO3 PEGylation of Protegrin-1 (PG-1) withN-m-PEG-Benzamide-p-Succinimidyl Carbonate (SBC)-30K

A stock solution of 1.2 mg/mL PG-1 was prepared in 2 mM HCl. To initiatea reaction, the PG-1 stock solution was brought to 25° C., a 15-foldmolar excess of SBC-30K lyophilized powder was with stirring followedimmediately with the addition of 1 M MES, pH 6, to give finalconcentrations of 1.0 mG/mL PG-1 (0.46 mM) and 50 mM MES. The reactionwas allowed to proceed for 20 minutes at 25° C. After 20 min, thereaction was quenched with 100 mM glycine in 100 mM HCl (10 mM finalglycine concentration) for 10 minutes. The reaction mixture was thendiluted with deionized sterile water until the conductivity was below1.0 mS/cm and the pH was adjusted to 4.0 with 1 M sodium acetate, pH4.5. diluted.

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using a column packed with SPHP media (GEHealthcare) on an AKTA Explorer 100 system (GE Healthcare). Buffer A was20 mM sodium acetate, pH 4.0, Buffer B was 20 mM sodium acetate and 1 MNaCl, pH 4.0. The AKTA Explorer plumbing system and SPHP column weresanitized with 1 M HCl and 1 M NaOH and the resin was equilibrated with10 column volumes Buffer A prior to sample loading. After loading, thecolumn was washed with 5 column volumes 100% A/0% B to remove un-reactedPEG reagent. PEGylated and nonPEGylated peptides were eluted using alinear gradient from 80% A/20% B to 0% A/100% B over 20 column volumeswith a linear flow rate of 90 cm/hour.

Fractions collected during cation exchange chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.1%TFA in water, and B, 0.05% TFA in acetonitrile. A Waters Symmetry C18column (4.6 mm×75 mm) was used with a flow rate of 1.0 ml/min and acolumn temperature of 50° C. Detection was carried out at 280 nm. Thecolumn was equilibrated in 0% B and conjugate separation was achievedusing the gradient timetable shown in T PRO3.1.

Step Time (min) % Mobile phase B 1 0.00 20.0 2 2.00 30.0 3 5.00 45.0 46.00 45.0 5 18.00 80.0 6 18.10 100.0 7 20.10 100.0 8 20.20 10.0

Fractions containing pure mono-[mPEG-SBC-30K]-[PG-1] as determined byRP-HPLC were pooled. Glacial acetic acid was added to the pooledfractions to a final concentration of 5% (v/v) and loaded onto a CG71Scolumn (Rohm Haas) for Endotoxin removal and buffer exchange. Prior tosample loading, the column had been washed with 5% acetic acid inacetonitrile and equilibrated with 5% acetic acid in water (v/v). Aftersample loading, the column was washed with 10 column volumes of 5%acetic acid and mono-[mPEG-SBC-30K]-[PG-1] was eluted with a linear0-100% gradient from 5% acetic acid to 5% acetic acid/95% Acetonitrile(v/v) over 10 column volumes. Fractions containing the conjugate asdetermined by analytical reversed phase HPLC, were pooled, lyophilizedand stored at −80° C.

A typical cation exchange SP-HP chromatogram is shown in FIG. PRO3.1.SDS-PAGE analysis of purified mono-[mPEG-SBC-30K]-[PG-1] is shown inFIG. PRO3.2 . RP-HPLC analysis of the purified conjugate is shown inFIG. PRO3.3, and MALDI-TOF analysis of the purified conjugate is shownin FIG. PRO3.4.

The purity of the mono-PEG-conjugate was >95% by SDS-PAGE and 96.6% byRP-HPLC analysis. The mass as determined by MALDI-TOF was within theexpected range.

FIG. PRO3.1 Typical cation exchange purification profile ofmono-[mPEG-SBC-30K]-[PG-1]. The mono-PEGylated conjugate and unreactedPEG are indicated. The blue line represents absorbance at 280 nm and thered line represents absorbance at 225 nm.

FIG. PRO3.2. SDS-PAGE (4-12% NuPage Bis-Tris, Invitrogen, with CoomassieBlue staining) of purified [mono]-[mPEG-SBC-30K-]-[Protegrin-1]. Lane 1,Mark12 MW markers; Lane 2, purified[mono]-[mPEG-SBC-30K-]-[Protegrin-1]. The apparent large molecularweight of the conjugate, about 97 kDa, is due to a slow mobility of themonomeric conjugate in the gel due to a high degree of PEG hydration.Impurities were not detected in Lane 2.

FIG. PRO3.3. Purity analysis of [mono]-[mPEG-SBC-30K-]-[Protegrin-1] byreversed phase HPLC. The purity of the purified conjugate is 96.6% % at280 nm. The peak with retention time at 15.3 min, is the SBC-30K PEGreagent and constitutes 3.4% of the sample. The peak at 4.5 minutes is acolumn-derived species and is not included in the sample

FIG. PRO3.4. MALDI-TOF spectrum of purified[mono]-[mPEG-SBC-30K-]-[Protegrin-1]. The major peak at 33.3 kDa iswithin the expected range for the molecular weight of[mono]-[mPEG-SBC-30K-]-[Protegrin-1]. The peak at 66.1 kDa, mayrepresent the singly charged conjugate dimer formed during MALDI-TOFanalysis.

E PRO4 PEGylation of Protegrin-1 (PG-1) with PEG-diButyrAldehyde-5KOHCCH₂CH₂CH₂—(OCH₂CH₂)₄—NH—COO-PEG-O—CO—NH—(OCH₂CH₂)₄—CH₂CH₂CH₂—CHO

Stock solutions of 8.0 mg/mL PG-1 and 200 mG/mL PEG-ButyAldehyde-5000were prepared in 2 mM HCl. To initiate a reaction, the two stocksolutions and a 1 M HEPES, pH 7.0, stock solution were brought to 25° C.and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 2.0 mg/mLPG-1, 50 mM HEPES and a 5-foldmolar excess of PEG-diButyrAldehyde-5K over PG-1, After 15 minutereaction, a 20-fold molar excess of NaBH₃CN over PEG was added and thereaction was allowed to continue for an additional 16 hours at 25° C.After 16 hr, 15 min total reaction time, the reaction was quenched with100 mM glycine in 100 mM HCl (10 mM final glycine concentration) for 1hour, after which glacial acetic acid was added to a final concentrationof 5% (v/v).

The PEGylated conjugate was purified from the reaction mixture byreversed phase chromatography using a column packed with CG71S media(Rohm Haas) on an AKTA Explorer 100 system (GE Healthcare). Buffer A was5% acetic acid/95% H₂O (v/v), and Buffer B was 5% acetic acid/95%acetonitrile (v/v). The AKTA Explorer plumbing system and the CG71Scolumn were sanitized with 1 M HCl and 1 M NaOH and the resin wasequilibrated with 10 column volumes Buffer A prior to sample loading.After loading, the column was washed with 6 CV of 80% Buffer A/20%Buffer B and the PEGylated and nonPEGylated peptides were eluted using alinear gradient from 80% A/20% B to 0% A/100% B over 15 column volumewith a linear flow rate of 90 cm/hour.

Fractions collected during CG71S reversed phase chromatography wereanalyzed using analytical reversed-phase HPLC. The mobile phases were:A, 0.1% TFA in water, and B, 0.05% TFA in acetonitrile. A WatersSymmetry C18 column (4.6 mm×75 mm) was used with a flow rate of 1.0ml/min and a column temperature of 50° C. Detection was carried out at280 nm. The column was equilibrated in 20% B and conjugate separationwas achieved using the gradient timetable shown in T PRO4.1.

Step Time (min) % Mobile phase B 1 0.00 20.0 2 2.00 30.0 3 5.00 45.0 46.00 45.0 5 18.00 80.0 6 18.10 100.0 7 20.10 100.0 8 20.20 10.0

Fractions containing pure[Protegrin-1]-[PEG-di-ButyrAldehyde-5K]-[Protegrin-1] as determined byRP-HPLC were pooled, lyophilized and stored at −80° C.

A typical reverse phase CG71S chromatogram is shown in FIG. PRO4.1.SDS-PAGE analysis of purified[Protegrin-1]-[PEG-di-ButyAldehyde-5K]-[Protegrin-1] is shown in FIG.PRO4.2. RP-HPLC analysis of the purified conjugate is shown in FIG.PRO4.3, and MALDI-TOF analysis of the purified conjugate is shown inFIG. PRO4.4.

The purity of the [Protegrin-1]-[PEG-di-ButyrAldehyde-5K]-[Protegrin-1]conjugate was >95% by SDS-PAGE analysis and 98.7% by RP-HPLC analysis.The mass as determined by MALDI-TOF was within the expected range.

FIG. PRO4.1 Typical reversed phase purification profile of[Protegrin-1]-[PEG-di-ButyrAldehyde-5K]-[Protegrin-1]. The conjugate andunreacted peptide are indicated. The blue line represents absorbance at280 nm.

FIG. PRO4.2. SDS-PAGE (12% NuPage Bis-Tris, Invitrogen, with CoomassieBlue staining) of purified[Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1]. Lane 1, Mark12 MWmarkers; Lane 2, 17 uG of purified[Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1], and Lane 3, 4 uGof purified [Protegrin-1]-[PEG-butyraldehyde-5K]-[Protegrin-1]. Theconjugate in Lane 2 migrates with a higher apparent molecular weightthan the conjugate in Lane 3, which may be a result of conjugateoligomer formation at higher concentrations. Impurities were notdetected (Lane 3).

FIG. PRO4.3. Purity analysis of[Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1] by reversed phaseHPLC. The purity of the purified conjugate is determined to be 98.7% at280 nm. The peaks at 2.2 and 4.5 min are column or solvent derivedspecies and are not included in the sample.

FIG. PRO4.4. MALDI-TOF spectrum of[Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1]. The peak at 9.6kDa is within the expected range for the molecular weight of theconjugate. The peak at 4.8 kDa may represent the molecular weight of thedoubly charged conjugate, and the peak at 7.5 kDa may represent aconjugate containing a single peptide. The peaks at 2292 Da and 2147 Daare due to an instrument filter effect.

Example PRO5 Conjugation of Protegrin-1 with Dextran Tetra EthyleneGlycol-Butyraldehyde 40K

Stock solutions of 0.3 mg/mL protegrin-1 and 55 mg/mL dextran tetraethylene glycol (TEG)-butyraldehyde 40K, both in 50 mM HEPES, pH 7.0,were prepared. To initiate a reaction, both stock solutions were broughtto 25° C. and then mixed in equal volumes. The reaction mixture wasstirred at 25° C. After 1 hour reaction, 100 μM sodium cyanoborohydride(final concentration) was added and the reaction was allowed to proceedfor an additional 4 hours.

The dextran-protegrin-1 conjugate was purified from the reaction mixtureby cation-exchange chromatography using CM Sepharose (GE Healthcare).Upon completion of the conjugation reaction, the reaction mixture wasdiluted 10-fold with water and loaded onto a column packed with CMSepharose resin. Buffer A was 10 mM HEPES, pH 7, and buffer B was 10 mMHEPES, pH 7, 1M NaCl. The resin was washed with buffer B andequilibrated with buffer A prior to sample loading. After loading, thecolumn was washed with 2 column volumes buffer A. Conjugated andnonconjugated peptides were eluted in a linear gradient of 0-100% bufferB in 10 column volumes at a flow rate of 7 mL/min (FIG. 1).

FIG. PRO5.1. Typical cation-exchange chromatography profile ofdextran-butryaldehyde-40K-protegrin-1. Fractions containing theconjugate are indicated in the box. The line represents absorbance at280 nm.

Fractions containing dextran-butyraldehyde-40K-protegrin-1 were pooled,dialyzed against water, lyophilized and stored at −80° C.

FIG. PRO5.2. SDS-PAGE analysis (4-12% gel) of purifieddextran-butryaldehyde-40K-protegrin-1. Dextran perturbs the gelmigration of the dextran-peptide conjugate and the conjugate's bandlocation is not indicative of its size. The marker (M) molecular weightunit is kDa.

E PRO6 PEGylation of Protegrin-1 with Bi-FunctionalmPEG-Butaraldehyde-2K

The conjugation reaction took place in an aqueous environment. 45.5 mgPG-1 was first dissolved into 9.1 mL PBS buffer to make a 5 mg/mL stocksolution. 100 mg (ALD)₂2K was then dissolved into 1 mL PBS to make a 100mg/mL stock solution. To initiate the conjugation, 0.867 mL (ALD)₂2Kstock solution was slowly mixed into 9.7 mL PG-1 stock solution drop bydrop under rapid stirring. 135 μl of 50 mg/mL sodium cyanoborohydride(BaBH₃CN) was added into the reaction mixture 30 min later to facilitatethe stable secondary amine linkage formation through reductiveamination. The BaBH₃CN to (ALD)₂2K molar ratio was set at 5 with BaBH₃CNin excess. The final net (ALD)₂2K (93% substitution) to PG-1 molar ratiowas at 2 with (ALD)₂2K in excess. The formation of PG-1-ButyrALD-2K-PG-1was confirmed by analytical RP-HPLC (T PRO6.1).

T PRO6.1: Analytical RP-HPLC method used to monitorPG-1-ButyrALD-2K-PG-1 production. Column: Waters Xbridge C18 5 μm4.6×160 mm. Mobile Phase A: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN. Columntemperature: 40° C. UV_(280nm) was used to follow the elution.

TIME (min) % Mobile phase B Flow rate (mL/min) 0.0 10 1.0 5 20 1.0 24 701.0 25 10 1.0

The (PG-1)-(ALD)₂2K-(PG-1) was purified by weak cation exchangechromatography using an AKTA Basic System. The reaction mixture wasfirst diluted 5 fold with buffer A [20 mM acetate, pH 4.0] to reducesample viscosity. The pH of the diluted sample was measured to be 4.0and the conductivity to be 4.8 mS/cm. The diluted sample mixture wasloaded onto a CM Sepharose FF column at 5 mL/min. After sample loading,the column was washed with 2CV 20% buffer B [20 mM acetate, 2 M NaCl, pH4.0]. The loading and washing steps were done manually. The resin waswashed until a flat UV_(280nm) absorption line was observed. A lineargradient elution was applied next from 20% to 60% buffer B within 10 CV.The flow rate was held constant at 5 ml/min during the whole process.The chromatogram of the elution step is shown in FIG. PRO6.1.

FIG. PRO6.1: PG-1 and (ALD)₂2K conjugates purification with CM SepharoseFF resin. The UV_(280nm) absorption curve is shown in blue and buffer Bpercentage is shown in green. The conductivity is shown in gray. Peak Icontains mono-conjugate and peak II contains the desired di-conjugate.

The peak I and II fractions were analyzed by RP-HPLC (T PRO6.1). Thedesired product was found in peak II. Based on their high purities, peakII fractions 18 to 33 were pooled. The 210 mL purified(PG-1)-(ALD)₂2K-(PG-1) fraction pool was first centrifuged with a MWCO10,000 Centricon to a final volume of 20 mL. The NaCl concentration wasthen lowered to less than 50 mM by dilution with 20 mM acetate, pH 4.0buffer (final conductivity 3.8 mS/cm). The volume was reduced to lessthan 10 mL with a second centrifugation using a MWCO 10,000 Centricon.

The net peptide concentration in the concentrated (PG-1)-(ALD)₂2K-(PG-1)sample was measured to be 0.88 mg/mL by BCA. The conjugate's purity wasdetermined at 96.2% by RP-HPLC. The number-average molecular weight wascalculated to be 6888.2 Da by MALDI-TOF which is the expected mass ofthe di-conjugate. A final yield of 6.6 mg purified(PG-1)-(ALD)₂2K-(PG-1) was obtained.

FIG. PRO6.2: RP-HPLC analysis of (PG-1)-(ALD)₂2K-(PG-1).FIG. PRO6.3: MALDI analysis of (PG-1)-(ALD)₂2K-(PG-1). The 4753.6 Dapeak might be the residual mono-conjugate contaminant. The 2136 Da peakrepresents the free peptide. The peak areas do not correspond to therelative amounts of the species in the sample.

E PRO7 PEGylation of protegrin-1 with mPEG2-butaraldehyde-40K

The conjugation reaction took place in an aqueous environment. 12 mgprotegrin-1 (PG-1) was first dissolved into 1.2 mL PBS buffer to make a10 mg/mL stock solution. 1500 mg ALD-40K was dissolved into 15 mL 2 mMHCl to make a 100 mg/mL stock solution. To initiate the conjugation,11.4 mL ALD-40K stock solution was slowly mixed into 1.2 mL PG-1 stocksolution drop by drop under rapid stirring. 360 μL of 50 mg/mL sodiumcyanoborohydride (BaBH₃CN) was added into the reaction mixtureimmediately following PEG addition to facilitate the stable secondaryamine linkage formation through reductive amination. The BaBH₃CN toALD-40K molar ration was 10 with BaBH₃CN in excess. The net ALD-40K(99.5% purity) to PG-1 molar ratio was 5 with ALD-40K in excess. Thereaction was allowed to proceed for 46 h at 22° C. for completion. Theformation of ALD40K-PG-1 was confirmed by analytical RP-HPLC using themethod described in T PRO7.1.

TABLE PRO7.2 Analytical RP-HPLC method used to monitor ALD40K-PG-1production. Column: Waters Xbridge C18 5 μm 4.6 × 160 mm. Mobile PhaseA: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN. Column temperature: 40° C.UV_(280 nm) was used to follow the elution. TIME (min) % Mobile phase BFlow rate (mL/min) 0.0 10 1.0 5 20 1.0 24 70 1.0 25 10 1.0

The ALD40K-PG-1 was purified by SP Sepharose HP resin using an AKTABasic System. The reaction mixture was first diluted 5 fold with bufferA [20 mM MES, pH 6.0] to reduce sample viscosity. The pH of the dilutedsample was measured to be 6.0 and the conductivity to be 5.2 mS/cm. Thediluted sample mixture was loaded onto an SP Sepharose HP column at 5mL/min. After sample loading, the column was washed with 100% buffer A.The column was then sequentially washed with a six step gradient (50,100, 150, 200, 250 and 300 mM NaCl in 20 mM MES, pH 6.0 buffer]. Eachwash step was controlled manually and was started only when theUV_(280nm) absorbance was completely flat from the previous wash. Theflow rate was constant at 5 ml/min during the whole process. TheALD40K-PG-1, peak II, was eluted at 300 mM NaCl. The chromatogram of theloading and elution is shown in FIG. PRO7.1.

FIG. PRO7.1.1 and 7.1.2: ALD40K-PG-1 purification with SP Sepharose HPresin. The UV_(280nm) absorption curve is shown and buffer B percentageis shown. The conductivity is shown. The NaCl concentration in washingand eluting steps are labeled. The ALD40K-PG-1, peak II, was eluted at300 mM NaCl.

The eluted peaks were analyzed by analytical RP-HPLC (T PRO7.1) andSDS-PAGE (FIG. PRO7.2). The desired product, ALD40K-PG-1, was eluted inpeak II. Based on their high purities, peak II fractions 18 to 33 werepooled.

The purified ALD40K-PG-1 pool was concentrated with a MWCO 10,000Centricon. The final NaCl concentration was also lowered to 150 mM with20 mM IVIES, pH 6.0 buffer dilution. SDS-PAGE of the purified andconcentrated ALD40K-PG-1 is shown in FIG. PRO7.2. The net peptideconcentration in the final ALD40K-PG-1 preparation was measured to be0.8 mg/mL by BCA. The purity was determined at 96.2% by RP-HPLC (TPRO7.1 and FIG. PRO7.3). The number-average molecular weight wascalculated to be 41524 Da by MALDI-TOF, which corresponds to theexpected mass of the conjugate (FIG. PRO7.4). A final yield of 7.6 mgpurified ALD40K-PG-1 was obtained.

FIG. PRO7.3: RP-HPLC analysis of ALD40K-PG-1 (lot #YW-pgALD40K-01).FIG. PRO7.4: MALDI analysis of ALD40K-PG-1 (lot #YW-pgALD40K-01). The˜85 KDa peak is believed to represent the ALD40K-PG-1 conjugate dimerformed as an artifact during MALDI analysis. The dimer is not detectedby SDS-PAGE.

Example PRO5 [mono]-[4,7-CG-PEG2-FMOC-NHS-40K]-[protegrin-1]-175PEGylation of protegrin-1 with 4,7-CG-PEG2-FMOC-NHS-40K

4,7-CG-PEG2-FMOC-NHS-40K (CG-40K)

The conjugation reaction took place in an aqueous environment. 12 mgprotegrin-1 (PG-1) was first dissolved into 1.2 mL PBS buffer to make a10 mg/mL stock solution. 550 mg CG-40K was dissolved into 5.5 mL 2 mMHCl to make a 100 mg/mL stock solution. To initiate the conjugation, 5.0mL CG-40K stock solution was slowly mixed into 1.2 mL PG-1 stocksolution drop by drop under rapid stirring. 5.0 mL 10×PBS buffer wasadded into the reaction mixture to maintain a relatively neutral pHduring the reaction (measured at 6.8). The net active CG-40K (95%purity, 77.8% substitution percentage) to PG-1 molar ratio was 1.7 withCG-40K in excess. The reaction was allowed to proceed for 330 min at 22°C. and 12 h at 4° C. for completion. The formation of CG40K-PG wasconfirmed by analytical RP-HPLC (Table 1).

TABLE PRO8.3 Analytical RP-HPLC method used to monitor CG40K-PGproduction. Column: Waters Xbridge C18 5 μm 4.6 × 160 mm. Mobile PhaseA: 0.1% TFA/H₂O and B: 0.1% TFA/CH₃CN. Column temperature: 40° C.UV_(280 nm) was used to follow the elution. TIME (min) % Mobile phase BFlow rate (mL/min) 0.0 10 1.0 5 20 1.0 24 70 1.0 25 10 1.0

The CG40K-PG-1 was purified by SP Sepharose HP resin using an AKTA BasicSystem. The reaction mixture was first diluted 5 fold with buffer A [20mM acetate, pH 4.0] to reduce sample viscosity. The diluted samplemixture was loaded onto an SP HP column at 5 mL/min. After sampleloading, the column was washed with 100% buffer A until the UV_(280nm)absorbance was flat. The conjugate was then eluted with a lineargradient of 0 to 80% buffer B [20 mM acetate, 1 M NaCl, pH 4.0] within10 CV. The flow rate was constant at 5 ml/min during the whole process.The chromatogram of the loading and elution is shown in FIG. PRO8.1.

FIG. PRO8.1: CG40K-PG-1 purification with SP Sepharose HP resin. TheUV_(280nm) absorption curve is shown in blue and buffer B percentage isshown in green. The conductivity is shown in gray.

The CG40K-PG-1 fractions were analyzed by analytical RP-HPLC (TablePRO8.1). Based on their high purities, fractions 16 to 19 were pooled.The purified CG40K-PG-1 pool was concentrated with a MWCO 10,000Centricon. The final NaCl concentration was also lowered to 150 mM with20 mM acetate, pH 4.0 buffer dilution.

The net peptide concentration in the final CG40K-PG-1 preparation wasmeasured to be 1.33 mg/mL by BCA. The purity was determined at 99.8% byRP-HPLC (T PRO8.1 and FIG. PRO8.2). The number-average molecular weightwas calculated to be 44033 Da by MALDI-TOF (FIG. PRO8.3). A final yieldof 11.97 mg purified CG40K-PG-1 was obtained.

FIG. PRO8.2: RP-HPLC analysis of purified CG40K-PG-1. Analysisconditions are described in T PRO8.1.

FIG. PRO8.3: MALDI-TOF analysis of purified CG40K-PG-1. The ˜90 KDa peakis believed to represent the CG40K-PG-1 conjugate dimer formed as anartifact during MALDI analysis. The dimer is not detected by SDS-PAGE

E PRO9

Hemolysis assay. Approximately 10 mL of blood was drawn from one adultrat into Na Heparin tube and kept in ice until use. Red blood cells werewashed three times with 10 mL of cold DPBS ((−) CaCl₂ and (−) MgCl₂) andcollected by sequential centrifugation at 3,000 g for 5 min at 4° C.Pellets of red blood cells were resuspended with DPBS ((−) CaCl₂ and (−)MgCl₂) and the total volume was brought up to initial volume of blooddrawn. One mL of resuspended red blood cells was resuspended with 49 mLof DPBS ((−) CaCl₂ and (−) MgCl₂). Incubation mixture was prepared by400 fold dilution of stock solution of test compounds with final volumeof 800 μl. Final concentration of test compounds was equimolar to thatof respective unconjugated compounds. Hemolysis incubation was done at37° C. with mild agitation. For releasable conjugates, test compoundswere preincubated in 1×PBS at 37° C. prior to hemolysis assay.Incubation mixture was centrifuged at 3,000 g for 5 min at 4° C., andthe absorbance at 550 nm was read from supernatant. The percent ofhemolysis was calculated relative to the 100% hemolysis produced by0.25% Triton X-100.

Compounds Description Note PG1 Protegrin 1 (PG1) Samples wereCAC40K-40K-PG1 PG1 conjugate with a releasable preincubated linker(release t_(1/2) in 1XPBS in 1XPBS at 37° C. = ~19.6 hr) at 37° C. priorto CAC40K-fulvene PEG moiety of CAC40K-PG1 hemolysis assay.PG1-ButyrALD- PG1 conjugate with a stable PG1 linker Dextran-PG1 PG1conjugate with a stable linker 2 mM HCl Buffer control Matrix controlMatrix control TritonX-100 Detergent Positive control

For PG1, hemolytic effects were almost eliminated by PEG conjugationwith a stable linker (PG1-ButyrALD-PG1, Dextran-PG1), (FIG. PRO9.1).Percent hemolysis of rat red blood cells by PG1 was comparable toliterature data obtained from human red blood cell assay (FIG. PRO9.2).PG1-ButyrALD-5K-PG1 appeared to have little hemolytic activity. However,its hemolytic activity was significantly less than PG1. PG1 releasedfrom CAC-40K-PG1 exhibited hemolytic activity, however, hemolyticeffects from CAC-40K-PG1 that had been preincubated for 96 hr (=5 timesof its release t_(1/2), ˜19.6 hr) was 60-70% of that from PG1. This lossof activity appears to be mostly due to the degradation of PG1 duringpreincubation period since PG1 preincubated for 96 hr retained about 60%hemolytic activity compared to that from PG1 preincubated for 0 hr. PEGmoiety itself (CAC40K-fulvene, mPEG2-NHS 20K-gly, and mPEG-SMB 30K-gly)did not cause hemolysis.

FIG. PRO9.1. Hemolysis relative to the 100% hemolysis produced by 0.25%Triton X-100.FIG. PRO9.1. Hemolysis by PEG reagent controls.FIG. PRO9.3. Hemolysis at the maximum concentrationFIG. PRO9.4. Hemolytic activities of PG-1: Human red blood cells wereincubated with 0 to 100 μg/ml of PG-1 in PBS for 1 h at 37° C. (Tran Det al (2008) Antimicrob. Agents Chemother 52:944-953)

E PRO10 Pharmacokinetic Studies of the Protegrin Conjugates

Twenty one (21) adult male Sprague-Dawley rats with indwelling jugularvein and carotid artery catheters (JVC/CAC) (Charles River Labs,Hollister, Calif.) were utilized for this study. The weight range of theanimals was 313-346 grams. All animals were food fasted overnight. Priorto dosing the rats were weighed, the tails and cage cards were labeledfor identification and the doses were calculated. Anesthesia was inducedand maintained with 3.0-5.0% isoflurane. The JVC and CAC wereexternalized, flushed with HEP/saline (10 IU/mL HEP/mL saline), plugged,and labeled to identify the jugular vein and carotid artery The predosesample was collected from the JVC. When all of the animals had recoveredfrom anesthesia and the predose samples were processed, the animals weredosed, intravenously (IV) via the JVC using a 1 mL syringe containingthe appropriate test article, the dead volume of the catheter wasflushed with 0.9% saline to ensure the animals received the correctdose.

Following a single IV dose, blood samples were collected at 0 (pre-dosecollected as described above), 2, 10, 30, 60, 120, 240, 360 minutes forNKT-10503 (parent protegrin-1) group and 0 (pre-dose collected asdescribed above), 2, 10, 30, 120, 240, 480, 1440 (24 hrs) minutes forthe other groups via the carotid artery catheter and processed as statedin the protocol. Following the last collection point, the animals wereeuthanized. Bioanalytical analysis: analysis of the plasma samples wasconducted using non-validated LC-MS/MS methods.

Pharmacokinetic Analyses: Noncompartmental PK data analysis and reportpreparation was completed. PK analysis was performed using WinNonlin(Version 5.2, Mountain View, Calif.-94014). Concentrations in plasmathat were below LLOQ were replaced with zeros prior to generating Tablesand PK analysis. The following PK parameters were estimated using plasmaconcentration-time profile of each animal:

-   -   C₀ Extrapolated concentration to time “zero”    -   C_(max) Maximum (peak) concentration    -   AUC_(all) Area under the concentration-time from zero to time of        last concentration value    -   T_(1/2(Z)) Terminal elimination half-life    -   AUC_(inf) Area under the concentration-time from zero to time        infinity    -   T_(max) Time to reach maximum or peak concentration following        administration    -   CL Total body clearance    -   V_(z) Volume of distribution based on terminal phase    -   V_(ss) Volume of distribution at steady state    -   MRT_(last) Mean residence time to last observable concentration

Releasable-PEG:

FIG. PRO10.1 and PRO10.2 show the mean plasma concentration-timeprofiles for CG-PEG₂-FMOC-40K-PG-1 and CAC-PEG₂-FMOC-40K-PG-1, theircorresponding PEG-metabolite and released Protegrin-1. FIG. PRO10.3shows the released Protegrin-1 levels after the administration of thetwo releasable PEG constructs versus the level of Protegrin-1 given asnative protein at the same dose (mg/kg).Table PRO10.1 summarizes the PK parameters of protegrin-1 followingequivalent protein mass of 1.6 mg/kg administered intravenously intorats via, CG-PEG₂-FMOC-40K-PG-1, CACPEG ₂-FMOC-40K-PG-1 or nativeprotegrin-1.

TABLE PRO10.1 Comparative PK Parameters of Protegrin-1 and PEGconjugates Test C_(max) AUC_(INF) T_(max) Compound (ng/mL) T_(1/2) (hr)(ng · hr/mL) (hr) MRT_(last) (hr) Protegrin-1 2050 ± 601  0.35 ± 1160 ±351 0.03 0.52 ± 0.05 0.10 CAC-PEG₂- 222 ± 189 0.13* 31.0* 0.03 0.13 ±0.02 FMOC-40K- PG-1 CG-PEG₂-  285 ± 30.0 1.95* 655*   0.03 2.53 ± 0.60FMOC-40K- PG-1 *n − 2

Non-Releasable-PEG:

FIG. PRO10.4 shows the mean plasma concentration-time profiles forNKT-10502, NKT-10519 and NKT-531 observed in this study. Table 2summarizes the PK parameters of NKT-10502, NKT-10519 and NKT-531following equivalent protein mass of 1.6 mg/kg administeredintravenously into rats. Based on the observed data, NKT-10502 appearedto be declined slower than NKT-10519 and NKT-531.

TABLE PRO10.2 Comparative PK Parameters of Non-Releasable-PEGProtegrin-1 Conjugates Following Equivalent Protein Mass IntravenousAdministration to Sprague Dawley rats (Mean ± SD) Test C_(max) AUC_(INF)MRT_(last) CL V_(ss) Compound (ng/mL) T_(1/2) (hr) (ug · hr/mL) (hr)(mL/hr/kg) (mL/kg) Protegrin-1 2050 ± 601 0.35 ± 0.10 1.16 ± 0.351 0.52± 0.05  1470 ± 434  848 ± 360 mPEG2- 6110 ± 664 10.8 ± 3.0  52.7 ± 16.3 5.6 ± 0.83 32.9 ± 12.4 320 ± 131 40K-PG-1 PG-1- 6520 ± 679 7.3 ± 1.1 7.7± 1.09 1.7 ± 0.14  209 ± 27.3  432 ± 40.5 PEG-2K- PG-1 PG-1-  7550 ±1680  6.8 ± 0.65 11.9 ± 0.866 1.6 ± 0.16  135 ± 9.22  248 ± 22.1 PEG-5K-PG-1

E V1 V681-mPEG Conjugates (V681 Herein Refers to all V681-Like Peptides)

a) mPEG-N^(ter)-V681 Via mPEG-SPC

V681 peptide is prepared and purified according to standard automatedpeptide synthesis or recombinant techniques known to those skilled inthe art. An illustrative polymeric reagent, mPEG-SPC reagent,

‘SPC’ Polymer Reagent

is covalently attached to the N-terminus of V681, to provide aN^(ter)-conjugate form of the peptide. mPEG-SPC 20 kDa, stored at −20°C. under argon, is warmed to ambient temperature. The reaction isperformed at room temperature. About 3-5-fold molar excess of mPEG-SPC20 kDa reagent is used based upon absolute peptide content. The mPEG-SPCreagent is weighed into a glass vial containing a magnetic stirrer bar.A solution of V681 prepared in phosphate buffered saline, PBS, pH 7.4 isadded and the mixture is stirred using a magnetic stirrer until themPEG-SPC is fully dissolved. The stirring speed is reduced and thereaction is allowed to proceed to formation of conjugate product. Thereaction is optionally quenched to terminate the reaction. The pH of theconjugate solution at the end of the reaction is measured and furtheracidified by addition of 0.1M HCl, if necessary, to bring the pH of thefinal solution to about 5.5. The conjugate solution is then analyzed bySDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N^(ter)-V681conjugate formation.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

b) V681-C^(ter)-mPEG

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the C-terminus of V681, to provide a C^(ter)-conjugate formof the peptide. For coupling to the C-terminus, a protected V681 peptideis prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. mPEG-NH₂ 20 kDa,stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. About 5-fold molar excess ofmPEG-NH₂, PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphoniumhexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, basedupon absolute peptide content. The mPEG-NH₂, PyBOP, HOBt are weighedinto a glass vial containing a magnetic stirrer bar. A solution ofProt-V681 peptide is prepared in N,N-dimethylformamide is added and themixture is stirred using a magnetic stirrer until the mPEG-NH₂ is fullydissolved. The stirring speed is reduced and the reaction is allowed toproceed to formation of conjugate product. The conjugate solution isthen analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt-V681-C^(ter)-mPEG conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theV681-C^(ter)-mPEG conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

c) V681-Cys(S-mPEG)

mPEG-Maleimide is obtained having a molecular weight of 5 kDa and havingthe basic structure shown below:

mPEG-MAL, 5 kDa

V681, which has a thiol-containing cysteine residue, is dissolved inbuffer. To this peptide solution is added a 3-5 fold molar excess ofmPEG-MAL, 5 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of this peptide.

Using this same approach, other conjugates are prepared using mPEG-MALhaving other weight average molecular weights.

d) mPEG-N^(ter)-V681-Via mPEG-SMB

An mPEG-N-Hydroxysuccinimide is obtained having a molecular weight of 5kDa and having the basic structure shown below:

mPEG-Succinimidyl α-Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)

mPEG-SMB, 5 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of the peptide)of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot of astock V681 peptide solution and mixed well. After the addition of themPEG-SMB, the pH of the reaction mixture is determined and adjusted to6.7 to 6.8 using conventional techniques. To allow for coupling of themPEG-SMB to the peptide via an amide linkage, the reaction solution isstirred for several hours (e.g., 5 hours) at room temperature in thedark or stirred overnight at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction is quenched with a 20-fold molarexcess (with respect to the peptide) of Iris buffer.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan N-hydroxysuccinimide moiety.

e) V681-Glu(O-mPEG)

An illustrative polymeric reagent, mPEG-NH₂ reagent is covalentlyattached to the Glu residue of V681, to provide a Glu-conjugate form ofthe peptide. For coupling to the Glu residue, a protected V681 peptideis prepared and purified according to standard automated peptidesynthesis techniques known to those skilled in the art. Deprotection ofthe Glu(OBz) residue (H₂/Pd) yields the free-Glu carboxylate forsubsequent coupling. mPEG-NH₂ 20 kDa, stored at −20° C. under argon, iswarmed to ambient temperature. The reaction is performed at roomtemperature. A 5-fold molar excess of mPEG-NH₂, PyBOP(benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate),and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptidecontent. The mPEG-NH₂, PyBOP, HOBt are weighed into a glass vialcontaining a magnetic stirrer bar. A solution of Prot3-V681 peptide isprepared in N,N-dimethylformamide is added and the mixture is stirredusing a magnetic stirrer until the mPEG-NH₂ is fully dissolved. Thestirring speed is reduced and the reaction is allowed to proceed toformation of conjugate product. The conjugate solution is then analyzedby SDS-PAGE and RP-HPLC (C18) to determine the extent ofProt3-V681-(Glu-O-mPEG) conjugate formation. The remaining protectinggroups are removed under standard deprotection conditions to yield theV681-Glu(O-mPEG) conjugate.

Using this same approach, other conjugates are prepared using mPEGderivatives having other weight-average molecular weights that also bearan amino moiety.

E V2

PEGylation of V681(V13AD) with [mPEG2-NHS-20K]

A stock solution of 4 mg/mL V681(V13AD) was prepared in water. Thepeptide stock solution was diluted 1:1 in 50 mM sodium phosphate, pH7.4, resulting in a peptide concentration of 2 mg/mL. Immediately beforea PEGylation reaction was initiated, a 14 mg/mL stock solution ofmPEG2-NHS-20K was prepared in 2 mM HCl. This PEG reagent forms stablebonds with amine groups. To initiate a reaction, the PEG stock solutionand 2 mg/mL peptide solution were brought to 25° C. and then mixed inequal volumes. The reaction mixture was stirred for 1 hour at 25° C.after which the reaction was quenched with 100 mM glycine in 2 mM HCl(10 mM final glycine concentration).

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using SP Sepharose HP media (GEHealthcare). The resin was packed in an XK 26/10 column (GE). Buffer Awas 20 mM sodium phosphate buffer, pH 7.4, and Buffer B was 20 mM sodiumphosphate, 1M NaCl, pH 7.4. The resin was washed in buffer B andequilibrated in buffer A before sample loading. After loading, the resinwas washed in buffer A for 2 column volumes and the PEGylated andnonPEGylated peptides were eluted using a linear gradient of 0-100% B in10 column volumes at a flow rate of 5 mL/min.

Fractions collected during cation exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were: A, 0.1% TFA in waterand B, 0.85% TFA in acetonitrile. An Agilent Poroshell 300-SB-C8 columnwas used with a flow rate of 0.2 ml/min and a column temperature of 50°C. Detection was carried out at 280 nm. The column was equilibrated in0% B and conjugate separation was achieved using the gradient timetableshown in T V2.1.

TIME (MIN) % MOBILE PHASE A % MOBILE PHASE B 0.00 100.0 0.0 5.00 100.00.0 10 70.0 30.0 20.00 30.0 70.0 21 20.0 80.0 25 20.0 80.0 30 100.0 0.0

Fractions containing pure [mono]-[mPEG2-20K]-[V681(V13AD)] as determinedby RP-HPLC and SDS-PAGE were pooled and concentrated over a reversedphase CG71S column. The column was washed with 0.5% acetic acid inacetonitrile and equilibrated with 0.5% acetic acid before loading.After loading, the column was washed with 0.5% acetic acid and thePEGylated peptide was eluted with 0.5% acetic acid in acetonitrile. Thefractions containing pure mono-PEGylated peptide were collected,lyophilized and stored at −80° C.

A typical cation-exchange chromatogram is shown in FIG. V2.1. SDS-PAGEanalysis of V681(V13AD) and purified [mono]-[mPEG2-20K]-[V681(V13AD)]conjugate is shown in FIG. V2.2. RP-HPLC analysis of the purifiedconjugate is shown in FIG. V2.3, and MALDI-TOF analysis of the purifiedconjugate is shown in FIG. V2.4.

The purity of the mono-PEG-conjugate was >95% by SDS-PAGE analysisand >98% by RP-HPLC analysis. The mass as determined by MALDI-TOF waswithin the expected range.FIG. V2.1. Typical cation-exchange purification profile of[mPEG2-NHS-20K]-[V681(V13AD)]. The mono-PEGylated conjugate is indicatedin a gray box and labeled B5-C2. The di-PEGylated conjugate did not bindto the resin. The blue line represents absorbance at 280 nm and the redline represents absorbance at 215 nm.FIG. V2.2. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofV681(V13AD) PEGylation and purification on the SP ion-exchange column.FIG. V2.3. Purity analysis of [mono]-[mPEG2-NHS 20K]-[V681(V13AD)]conjugate by reverse phase HPLC. The purity of the purified conjugatewas determined to be NLT 95% at 280 nm. 1.0% of the sample eluted at15.9 min which corresponds to the nonPEGylated peptide. The peak at 13minutes contains column-derived species and is not specific to thesample.FIG. V2.4. MALDI-TOF spectra for [mono]-[mPEG2-NHS 20K]-[V681(V13AD)].The major peak at 20.2 kD represents the molecular weight of monomeric[mono]-[mPEG2-NHS 20K]-[V681(V13AD)] conjugate.

E V3 PEGylation of V681(V13AD) with [mPEG-SMB-30K]

A stock solution of 4 mg/mL V681(V13AD) was prepared in water. Thepeptide stock solution was diluted 1:1 in 50 mM sodium phosphate, pH7.4, resulting in a peptide concentration of 2 mg/mL. Immediately beforea PEGylation reaction was initiated, a 20 mg/mL stock solution ofmPEG-SMB-30K was prepared in 2 mM HCl. This PEG reagent forms stablebonds with amine groups. To initiate a reaction, the PEG stock solutionand 2 mg/mL peptide solution were brought to 25° C. and then mixed inequal volumes. The reaction mixture was stirred for 1 hour at 25° C.after which the reaction was quenched with 100 mM glycine in 2 mM HCl(10 mM final glycine concentration).

The mono-PEGylated conjugate was purified from the reaction mixture bycation exchange chromatography using SP Sepharose HP media (GEHealthcare). The resin was packed in an XK 26/10 column (GE). Buffer Awas 20 mM sodium phosphate buffer, pH 7.4, and Buffer B was 20 mM sodiumphosphate, 1M NaCl, pH 7.4. The resin was washed in buffer B andequilibrated in buffer A before sample loading. After loading, the resinwas washed in buffer A for 2 column volumes and the PEGylated andnonPEGylated peptides were eluted using a linear gradient of 0-100% B in10 column volumes at a flow rate of 5 mL/min.

Fractions collected during cation exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were: A, 0.1% TFA in waterand B, 0.85% TFA in acetonitrile. An Agilent Poroshell 300-SB-C8 columnwas used with a flow rate of 0.2 ml/min and a column temperature of 50°C. Detection was carried out at 280 nm. The column was equilibrated in0% B and conjugate separation was achieved using the gradient timetableshown in T V3.1.

TIME (MIN) % MOBILE PHASE A % MOBILE PHASE B 0.00 100.0 0.0 5.00 100.00.0 10 70.0 30.0 20.00 30.0 70.0 21 20.0 80.0 25 20.0 80.0 30 100.0 0.0

Fractions containing pure [mono]-[mPEG-SMB-30K]-[V681(V13AD)] asdetermined by RP-HPLC and SDS-PAGE were pooled and concentrated over areversed phase CG71S column. The column was washed with 0.5% acetic acidin acetonitrile and equilibrated with 0.5% acetic acid before loading.After loading, the column was washed with 0.5% acetic acid and thePEGylated peptide was eluted with 0.5% acetic acid in acetonitrile. Thefractions containing pure PEGylated peptide were collected, lyophilizedand stored at −80° C.

A typical cation-exchange chromatogram is shown in FIG. V3.1. SDS-PAGEanalysis of V681(V13AD) and purified [mono]-[mPEG-SMB-30K]-[V681(V13AD)]conjugate is shown in FIG. V3.2. RP-HPLC analysis of the purifiedconjugate is shown in FIG. V3.3, and MALDI-TOF analysis of the purifiedconjugate is shown in FIG. V3.4.

FIG. V3.1. Typical cation-exchange purification profile of[mPEG-SMB-30K]-[V681(V13AD)]. The mono-PEGylated conjugate is indicatedin B5-C2. The di-PEGylated conjugate did not bind to the resin. The blueline represents absorbance at 280 nm and the red line representsabsorbance at 215 nm.FIG. V3.2. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofV681(V13AD) PEGylation and purification on the SP ion-exchange column.FIG. V3.3. Purity analysis of [mono]-[mPEG-SMB-30K]-[V681(V13AD)]conjugate by reverse phase HPLC. The purity of the purified conjugatewas determined to be NLT 95% at 280 nm. 1.0% of the sample eluted at15.9 min which corresponds to the nonPEGylated peptide. The peak at 13minutes contains column-derived species and is not specific to thesample.FIG. V3.4. MALDI-TOF spectra for [mono]-[mPEG-SMB 30K]-[V681(V13AD)].The major peak at 33.9 KDa represents the molecular weight of monomeric[mono]-[mPEG-SMB 30K]-[V681(V13AD)] conjugate.

E V4

Compare pharmacokinetics of non-releasable SMB-30K-V681 (V13AD)), andNHS-20K-V681 (V13AD)), with (parent V681 (V13AD)).

Study Design and Conduct

Procedure: Nine (9) adult male Sprague-Dawley rats with indwellingjugular vein and carotid artery catheters (JVC/CAC) (Charles River Labs,Hollister, Calif.) were utilized for this study. The weight range of theanimals was 311-346 grams. All animals were food fasted overnight. Priorto dosing the rats were weighed, the tails and cage cards were labeledfor identification and the doses were calculated. Anesthesia was inducedand maintained with 3.0-5.0% isoflurane. The JVC and CAC wereexternalized, flushed with HEP/saline (10 IU/mL HEP/mL saline), plugged,and labeled to identify the jugular vein and carotid artery. The predosesample was collected from the JVC. When all of the animals had recoveredfrom anesthesia and the predose samples were processed, the animals weredosed, intravenously (IV) via the JVC using a 1 mL syringe containingthe appropriate test article, the dead volume of the catheter wasflushed with 0.9% saline to ensure the animals received the correctdose.

Following a single IV dose, blood samples were collected into EDTAmicrotainers containing 75 μL of protease inhibitor cocktail at 0(pre-dose collected as described above), 2, 10, 30 minutes and at 1, 2,4, 8, 24 hrs via the carotid artery catheter and processed as stated inthe protocol. Following the last collection point, the animals wereeuthanized.

Bioanalytical Analysis:

Pharmacokinetic Analyses: Noncompartmental PK data analysis and reportpreparation was completed by Research Biology at Nektar Therapeutics atSan Carlos, Calif. Individual plasma concentration data are listed andsummarized in Appendix A1.1-1.3. PK analysis was performed usingWinNonlin (Version 5.2, Mountain View, Calif.-94014). Concentrations inplasma that were below LLOQ were replaced with zeros prior to generatingTables and PK analysis. The following PK parameters were estimated usingplasma concentration-time profile of each animal:

-   -   C₀ Extrapolated concentration to time “zero”    -   C_(max) Maximum (peak) concentration    -   AUC_(all) Area under the concentration-time from zero to time of        last concentration value    -   T_(1/2(Z)) Terminal elimination half-life    -   AUC_(inf) Area under the concentration-time from zero to time        infinity    -   T_(max) Time to reach maximum or peak concentration following        administration    -   CL Total body clearance    -   V_(z) Volume of distribution based on terminal phase    -   V_(ss) Volume of distribution at steady state    -   MRT Mean residence time

FIG. V4.1 shows the mean plasma concentration-time profiles for V681(V13AD), SMB-30K-V681 (V13AD), and NHS-20K-V681 (V13AD), observed inthis study. Both SMB-30K-V681 (V13AD), and NHS-20K-V681 (V13AD) arenon-releasable PEGylated conjugates and were shown to have slowerdeclining profiles and higher systemic exposure compared to the nativeV681 (V13AD). A very low but detectable level of V681 (V13AD) wasobserved in the first few timepoints (2-30 minutes) after SMB-30K-V681(V13AD), and NHS-20K-V681 (V13AD), administration.

T V4.1 summarizes the PK parameters of V681 (V13AD), SMB-30K-V681(V13AD), and NHS-20K-V681 (V13AD) following equivalent protein mass of1.0 mg/kg administered intravenously into rats. Based on the observeddata, SMB-30K-V681 (V13AD), and NHS-20K-V681 (V13AD), had significantlonger mean t_(1/2) compared with V681 (V13AD). The mean AUC ofSMB-30K-V681 (V13AD), and NHS-20K-V681 (V13AD), were 123 and 24 times ofV681 (V13AD), respectively.

TABLE V4.1 AUC_(INF) CL V_(ss) Compound C_(max) (ng/mL) T_(1/2) (hr) (μg· hr/mL) MRT (hr) (mL/hr/kg) (mL/kg) V681 16500 ± 9670 0.61 ± 0.39 7.21± 1.43 0.33 ± 0.08 142 ± 26  47.7 ± 18.0 (V13AD) SMB-30K- 4380 ± 50526.6 ± 12.5 158 ± 43  38.3 ± 18.2 6.6 ± 1.6 235 ± 50  V681 (V13AD)NHS-20K- 5210 ± 211 5.8 ± 1.0 53.0 ± 1.1  7.6 ± 1.1 18.9 ± 0.39  143 ±23.7 V681 (V13AD)

E V5

Hemolysis assay. Approximately 10 mL of blood was drawn from one adultrat into Na Heparin tube and kept in ice until use. Red blood cells werewashed three times with 10 mL of cold DPBS ((−) CaCl₂ and (−) MgCl₂) andcollected by sequential centrifugation at 3,000 g for 5 min at 4° C.Pellets of red blood cells were resuspended with DPBS ((−) CaCl₂ and (−)MgCl₂) and the total volume was brought up to initial volume of blooddrawn. One mL of resuspended red blood cells was resuspended with 49 mLof DPBS ((−) CaCl₂ and (−) MgCl₂). Incubation mixture was prepared by400 fold dilution of stock solution of test compounds with final volumeof 800 μl. Final concentration of test compounds was equimolar to thatof respective unconjugated compounds. Hemolysis incubation was done at37° C. with mild agitation.

For releasable conjugates, test compounds were preincubated in 1×PBS at37° C. prior to hemolysis assay. Incubation mixture was centrifuged at3,000 g for 5 min at 4° C., and the absorbance at 550 nm was read fromsupernatant. The percent of hemolysis was calculated relative to the100% hemolysis produced by 0.25% Triton X-100.

Compounds Description Note V681(V13AD) Native peptide mPEG2-NHS 20K-V681(V13AD) conjugate with a V681(V13AD) stable linker mPEG-SMB 30K-V681(V13AD) conjugate with a V681(V13AD) stable linker mPEG2-NHS 20K-glyPEG moiety of mPEG2-NHS 20K-V681(V13AD) mPEG-SMB 30K-gly PEG moiety ofmPEG-SMB 30K-V681(V13AD) V681(V13AD)desA12 2 mM HCl Buffer controlMatrix control Matrix control TritonX-100 Detergent Positive controlFor V681(V13AD), hemolytic effects were almost eliminated by PEGconjugation with a stable linker mPEG2-NHS 20K-V681(V13AD), and mPEG-SMB30K-V681(V13AD)] (FIG. V5.1).

Example C-PEP2 PEGylation of C-Peptide(S20C) with [mPEG-ru-MAL-30K]

A stock solution of 4 mg/mL C-peptide(S20C) was prepared in water. Thepeptide stock solution was diluted 1:1 in 20 mM sodium citrate, pH 5,resulting in a peptide concentration of 2 mg/mL. Immediately before aPEGylation reaction was initiated, an 80 mg/mL stock solution ofmPEG-ru-MAL-30K was prepared in 2 mM HCl. This PEG reagent forms stablebonds with thiol groups. To initiate a reaction, the PEG stock solutionand 2 mg/mL peptide solution were brought to 25° C. and then mixed inequal volumes. The reaction mixture was stirred for 16 hours at 25° C.

The mono-PEGylated conjugate was purified from the reaction mixture byanion exchange chromatography using Q HP Sepharose HP media (GEHealthcare). The resin was packed in an XK 16/10 column (GE). Buffer Awas 20 mM HEPES, pH 7.0, and Buffer B was 20 mM HEPES, pH 7.0, 1M NaCl.The resin was washed in buffer B and equilibrated in buffer A prior tosample loading. After loading, the resin was washed with 2 columnvolumes buffer A and the PEGylated and nonPEGylated peptides were elutedusing a linear gradient of 0-100% B in 5 column volumes at a flow rateof 3 mL/min.

Fractions collected during anion exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were: A, 0.1% TFA in waterand B, 0.85% TFA in acetonitrile. An Agilent Poroshell 300-SB-C8 columnwas used with a flow rate of 0.2 ml/min and a column temperature of 50°C. Detection was carried out at 215 nm. The column was equilibrated in0% B and conjugate separation was achieved using the gradient timetableshown in T-PEP2.1.

TIME (MIN) % MOBILE PHASE A % MOBILE PHASE B 0.00 100.0 0.0 5.00 70.030.0 15.00 40.0 60.0 20.00 20.0 80.0

T-PEP2.1. RP-HPLC Timetable

Fractions containing pure [mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)] asdetermined by analytical RP-HPLC were pooled and concentrated over areversed phase CG71S column. The column was washed with 0.5% acetic acidin acetonitrile and equilibrated with 0.5% acetic acid prior to sampleloading. After loading, the column was washed with 0.5% acetic acid andthe PEGylated peptide was eluted with 0.5% acetic acid in acetonitrile.Fractions containing PEGylated peptide were collected, lyophilized andstored at −80° C.

A typical anion-exchange chromatogram is shown in FIG. 1.1. RP-HPLCanalysis of purified [mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)] is shownin FIG. 1.2 and MALDI-TOF analysis of the purified conjugate is shown inFIG. 1.3.

The purity of the mono-PEG-conjugate was >98% by RP-HPLC analysis. Themass as determined by MALDI-TOF was within the expected range.

FIG. C-PEP 2.1. Typical anion-exchange chromatography profile of[[mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)]. The mono-PEGylatedconjugate is indicated in the grey box. The blue line representsabsorbance at 215 nm.

FIG. C-PEP 2.2. Purity analysis of[[mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)] by reversed phase HPLC. Thepurity of the purified conjugate was determined to be NLT 95% at 215 nm.

FIG. C-PEP2.3. MALDI-TOF spectrum for[mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)]. The major peak at 34.7 kDrepresents the molecular weight of monomeric[mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)].

Example C-PEP3 PEGylation of C-Peptide(S20C) with[mPEG-Butyraldehyde-30K]

A stock solution of 4 mg/mL C-peptide(S20C) was prepared in water. Thepeptide stock solution was diluted 1:1 in 20 mM sodium citrate, pH 6,resulting in a peptide concentration of 2 mg/mL. Immediately before aPEGylation reaction was initiated, a 60 mg/mL stock solution ofmPEG-Butyraldehyde-30K was prepared in 2 mM HCl. This PEG reagent formsstable bonds with amine groups. To initiate a reaction, the PEG stocksolution and 2 mg/mL peptide solution were brought to 25° C. and thenmixed in equal volumes. The reaction mixture was stirred for 1 hour at25° C. After 1 hour, 10 mM sodium cyanoborohydride (final concentration)was added and the reaction was mixed for a further 16 hours at 25° C.After 16 hours, 100 mM glycine in 2 mM HCl was added (10 mM finalglycine concentration).

The mono-PEGylated conjugate was purified from the reaction mixture byanion exchange chromatography using Q HP Sepharose HP media (GEHealthcare). The resin was packed in an XK 16/10 column (GE). Buffer Awas 20 mM HEPES, pH 7.0, and Buffer B was 20 mM HEPES, pH 7.0, 1M NaCl.The resin was washed in buffer B and equilibrated in buffer A prior tosample loading. After loading, the resin was washed with 2 columnvolumes buffer A and the PEGylated and nonPEGylated peptides were elutedusing a linear gradient of 0-100% B in 5 column volumes at a flow rateof 5 mL/min.

Fractions collected during anion exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were: A, 0.1% TFA in waterand B, 0.85% TFA in acetonitrile. An Agilent Poroshell 300-SB-C8 columnwas used with a flow rate of 0.2 ml/min and a column temperature of 50°C. Detection was carried out at 215 nm. The column was equilibrated in0% B and conjugate separation was achieved using the gradient timetableshown in T-PEP3.1.

TIME (MIN) % MOBILE PHASE A % MOBILE PHASE B 0.00 100.0 0.0 5.00 100.00.0 10.00 70.0 30.0 20.00 30.0 70.0 21.00 20.0 80.0 25.00 20.0 80.0

T-PEP3.1. RP-HPLC Timetable

Fractions containing pure[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)] as determined byanalytical RP-HPLC were pooled and concentrated over a reversed phaseCG71S column. The column was washed with 0.5% acetic acid inacetonitrile and equilibrated with 0.5% acetic acid prior to sampleloading. After loading, the column was washed with 0.5% acetic acid andthe PEGylated peptide was eluted with 0.5% acetic acid in acetonitrile.Fractions containing PEGylated peptide were collected, lyophilized andstored at −80° C.

A typical anion-exchange chromatogram is shown in FIG. 1.1. RP-HPLCanalysis of purified [mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)]is shown in FIG. 1.2 and MALDI-TOF analysis of the purified conjugate isshown in FIG. 1.3.

The purity of the mono-PEG-conjugate was >98% by RP-HPLC analysis. Themass as determined by MALDI-TOF was within the expected range.

FIG. C-PEP3.1. Typical anion-exchange chromatography profile of[[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)]. The mono-PEGylatedconjugate is indicated in the grey box. The blue line representsabsorbance at 215 nm.

FIG. C-PEP3.2. Purity analysis of[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)] by reversed phaseHPLC. The purity of the purified conjugate was determined to be NLT 95%at 215 nm.

FIG. C-PEP3.3. MALDI-TOF spectrum for[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)]. The major peak at33.8 kD represents the molecular weight of monomeric[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)].

Example C-PEP4 PEGylation of C-Peptide(S20C) with [C2-PEG2-FMOC-NHS-40K]

A stock solution of 2 mg/mL C-peptide(S20C) was prepared in 20 mM HCl.Immediately before a PEGylation reaction was initiated, a 56 mg/mL stocksolution of C2-PEG2-FMOC-NHS-40K was prepared in 20 mM HCl. This PEGreagent forms reversible bonds with amine groups. To initiate areaction, the two stock solutions were brought to 25° C. and then mixedin equal volumes. 1M sodium bicarbonate, pH 10.0, was immediately added(32 mM final concentration) and the reaction mixture was mixed for 10minutes at 25° C. The reaction was quenched and the pH was lowered to6.0 by the addition of 100 mM glycine in 100 mM HCl (10 mM final glycineconcentration). After quenching, the mixture was diluted 4-fold with 10mM ammonium acetate, pH 5.

The mono-PEGylated conjugate was purified from the reaction mixture byanion exchange chromatography using Q HP Sepharose media (GEHealthcare). The resin was packed in an XK 26/10 column (GE). Buffer Awas 10 mM ammonium acetate, pH 5, and Buffer B was 10 mM ammoniumacetate, pH 5, 1M NaCl. The resin was washed in buffer B andequilibrated in buffer A prior to sample loading. After loading, theresin was washed with 2 column volumes buffer A and the PEGylated andnonPEGylated peptides were eluted using a linear gradient of 0-100% B in10 column volumes at a flow rate of 8 mL/min.

Fractions collected during anion exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were: A, 0.1% TFA in waterand B, 0.85% TFA in acetonitrile. An Agilent Poroshell 300-SB-C8 columnwas used with a flow rate of 0.2 ml/min and a column temperature of 50°C. Detection was carried out at 215 nm and 313 nm. The column wasequilibrated in 0% B and conjugate separation was achieved using thegradient timetable shown in T-PEP4.1.

TIME (MIN) % MOBILE PHASE A % MOBILE PHASE B 0.00 100.0 0.0 5.00 100.00.0 10.00 70.0 30.0 20.00 30.0 70.0 21.00 20.0 80.0 25.00 20.0 80.030.00 100.0 0.0

Fractions containing pure [mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)] asdetermined by analytical RP-HPLC were pooled and concentrated over areversed phase CG71S column. The column was washed with 0.5% acetic acidin acetonitrile and equilibrated with 0.5% acetic acid prior to sampleloading. After loading, the column was washed with 0.5% acetic acid andthe PEGylated peptide was eluted with 0.5% acetic acid in acetonitrile.Fractions containing PEGylated peptide fractions were collected,lyophilized and stored at −80° C.

A typical anion-exchange chromatogram is shown in FIG. C-PEP4.1. RP-HPLCanalysis of [mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)] is shown in FIG.C-PEP4.2, and MALDI-TOF analysis of the purified conjugate is shown inFIG. C-PEP4.3.

The purity of the mono-PEG-conjugate was >98% by RP-HPLC analysis. Themass as determined by MALDI-TOF was within the expected range.

FIG. C-PEP4.1. Typical anion-exchange chromatography profile of[mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)]. The mono-PEGylatedconjugate is indicated in the grey box. The blue line representsabsorbance at 215 nm and the red line represents absorbance at 313 nm.FIG. C-PEP4.2. Purity analysis of[[mono]-[C2-PEG2-FMOC-40K]C-peptide(S20C)] by reversed phase HPLC.FIG. C-PEP4.3. MALDI-TOF spectrum for[mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)]. The major peak at 44.0 kDrepresents the molecular weight of monomeric[mono]-[mPEG-CAC-PEG2-FMOC-40K]-[C-peptide(S20C)].

Example C-PEP5 PEGylation of C-Peptide(S20C) with[CAC-PEG2-FMOC-NHS-40K]

A stock solution of 4 mg/mL C-peptide(S20C) was prepared in water. Thepeptide stock solution was diluted 1:1 in 1M HEPES, pH 7.0, resulting ina peptide concentration of 2 mg/mL. Immediately before a PEGylationreaction was initiated, a 128 mg/mL stock solution ofCAC-PEG2-FMOC-NHS-40K was prepared in 2 mM HCl. This PEG reagent formsreversible bonds with amine and thiol groups. To initiate a reaction,the PEG stock solution and 2 mg/mL peptide solution were brought to 25°C. and then mixed in equal volumes. The reaction mixture was stirred for3 hours at 25° C. After 3 hours, 100 mM Glycine in 2 mM HCl was added(10 mM final glycine concentration).

The mono-PEGylated conjugate was purified from the reaction mixture byanion exchange chromatography using Q HP Sepharose HP media (GEHealthcare). The resin was packed in an XK 26/10 column (GE). Buffer Awas 10 mM HEPES, pH 7.0, and Buffer B was 10 mM HEPES, pH 7.0, 1M NaCl.The resin was washed in buffer B and equilibrated in buffer A prior tosample loading. After loading, the resin was washed with 2 columnvolumes buffer A and the PEGylated and nonPEGylated peptides were elutedusing a linear gradient of 0-100% B in 10 column volumes at a flow rateof 7 mL/min.

Fractions collected during anion exchange chromatography were analyzedusing reversed-phase HPLC. The mobile phases were: A, 0.1% TFA in waterand B, 0.85% TFA in acetonitrile. An Agilent Poroshell 300-SB-C8 columnwas used with a flow rate of 0.2 ml/min and a column temperature of 50°C. Detection was carried out at 215 nm and 313 nm. The column wasequilibrated in 0% B and conjugate separation was achieved using thegradient timetable shown in T-PEP5.1.

TIME (MIN) % MOBILE PHASE A % MOBILE PHASE B 0.00 100.0 0.0 5.00 100.00.0 10.00 70.0 30.0 20.00 30.0 70.0 21.00 20.0 80.0 25.00 20.0 80.0

Fractions containing pure [mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20)] asdetermined by analytical RP-HPLC were pooled and concentrated over areversed phase CG71S column. The column was washed with 0.5% acetic acidin acetonitrile and equilibrated with 0.5% acetic acid prior to loading.After loading, the column was washed with 0.5% acetic acid and thePEGylated peptide was eluted with 0.5% acetic acid in acetonitrile.Fractions containing PEGylated peptide were collected, lyophilized andstored at −80° C.

A typical anion-exchange chromatogram is shown in FIG. C-PEP5.1. RP-HPLCanalysis of [mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20C)] is shown inFIG. C-PEP5.2 and MALDI-TOF analysis of the purified conjugate is shownin FIG. C-PEP5.3.

The purity of the mono-PEG-conjugate was >98% by RP-HPLC analysis. Themass as determined by MALDI-TOF was within the expected range.

FIG. C-PEP5.1. Typical anion-exchange purification profile of[[mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20C)]. The mono-PEGylatedconjugate and nonPEGylated peptide (Free) are indicated in grey boxes.The blue line represents absorbance at 215 nm and the purple linerepresents absorbance at 313 nm.

FIG. C-PEP5.2. Purity analysis of[mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20C)] by reversed phase HPLC. NoFree peptide was detected. The first peak contains monoconjugatedpeptide and the second peak contains primarily di-conjugated peptide inwhich the N-terminal amine and cysteine thiol groups are bothconjugated.

Example C-PEP6 Conjugation of C-Peptide(S20C) with DextranTetraethyleneglycol-butyraldehyde 40K

Stock solutions of 2 mg/mL C-peptide(S20C) and 200 mg/mL dextran tetraethylene glycol (TEG)-butyraldehyde 40K, both in 500 mM HEPES, pH 7.0,were prepared. To initiate a reaction, both stock solutions were broughtto 25° C. and then mixed in equal volumes. The reaction mixture wasstirred at 25° C. After 1 hour reaction, 10 mM sodium cyanoborohydride(final concentration) was added and the reaction was allowed to proceedfor an additional 16 hours.

The dextran-C-peptide(S20C) conjugate was purified from the reactionmixture by anion-exchange chromatography using Q HP Sepharose resin (GEHealthcare). Upon completion of the conjugation reaction, the reactionmixture was diluted 2-fold with water and loaded onto a column packedwith the Sepharose resin. Buffer A was 10 mM HEPES, pH 7.0, and buffer Bwas 10 mM HEPES, pH 7.0, 1.0 M NaCl. The resin was washed with buffer Band equilibrated with buffer A prior to sample loading. After loading,the column was washed with 2 CV buffer A. Conjugated and nonconjugatedpeptides were eluted in a linear gradient of 0-100% buffer B in 10 CV ata flow rate of 8 mL/min.

Fraction II collected during chromatography with Q HP Sepharose wasdiluted 10-fold with water and re-loaded onto the Q column in order toconcentrate the conjugate. The conjugate was eluted with 100% buffer B.

Fractions collected during both anion exchange chromatography runs wereanalyzed using reversed-phase HPLC. The mobile phases were: A, 0.1% TFAin water and B, 0.85% TFA in acetonitrile. An Agilent Poroshell300-SB-C8 column was used with a flow rate of 0.2 ml/min and a columntemperature of 50° C. Detection was carried out at 215 nm. The columnwas equilibrated in 0% B and conjugate separation was achieved using thegradient timetable shown in T-PEP6.1.

Time (min) % Mobile phase A % Mobile phase B 0.00 100.0 0.0 5.00 70.030.0 15.00 40.0 60.0 20.00 20.0 80.0

The concentrated purified conjugate collected from the second anionexchange chromatography run was dialyzed against water and frozen at−80° C.

Typical anion-exchange chromatograms of the reaction mixture andFraction II are shown in FIG. C-PEP6.1 and FIG. C-PEP6.2, respectively.RP-HPLC analysis of purified [mono]-[Dextran-40K]-[C-peptide(S20C)] isshown in FIG. C-PEP6.3 and MALDI-TOF analysis of the purified conjugateis shown in FIG. C-PEP6.4.

The purity of the mono-dextran conjugate was >93% by RP-HPLC analysis.The mass as determined by MALDI-TOF was within the expected range.

FIG. C-PEP6.1 Typical anion-exchange chromatography profile ofdextran-butryaldehyde-40K-C-peptide(S20C). Fractions containing theconjugate are indicated in box II. The blue line represents absorbanceat 215 nm.FIG. C-PEP6.2. Concentration of fraction II from the anion-exchangechromatogram shown in FIG. 1.1 by a second anion-exchange chromatographyrun. The blue line represents absorbance at 215 nm.FIG. C-PEP6.3. Purity analysis of [[mono]-[Dextran-40K]C-peptide(S20C)]by reversed phase HPLC. The purity of the purified conjugate wasdetermined to be NLT 93% at 215 nm.FIG. C-PEP6.4. MALDI-TOF spectrum for[mono]-[Dextran-40K]-[C-peptide(S20C)]. The peaks at 43.2 kDa and 22.0kDa agree with molecular weights of the single and double charged formsof the conjugated peptide.

E OGF2 PEGylation of Opioid Growth Factor (OGF) with[mPEG2-CAC-FMOC-NHS-40K]

Stock solutions of 2.0 mg/mL OGF and 200 mG/mL mPEG2-CAC-FMOC-NHS-40Kwere prepared in 2 mM HCl. To initiate a reaction, the two stocksolutions and a 0.5 M MES, pH 6.0, stock solution were brought to 25° C.and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.25 mg/mL OGF (2.2 mM), 20 mM MES and a1.25-fold molar excess of OGF over mPEG2-CAC-FMOC-NHS-40K. After 3 hoursat 25° C. the reaction was quenched with 100 mM glycine in 100 mM HCl(10 mM final glycine concentration) for 10 minutes. The quenchedreaction mixture was diluted with deionized sterile H₂O until theconductivity of the diluted reaction mixture was below 0.5 mS/cm, andthe pH was then adjusted to 6.0 with 1 M NaHCO₃/Na₂CO₃, pH 10.0.

The mono-PEGylated conjugate was purified from the diluted reactionmixture by anion exchange chromatography using a column packed with Q-HPmedia (GE Healthcare) and reversed phase chromatography using a columnpacked with CG17S media (Rohm Haas) on an AKTA Explorer 100 system (GEHealthcare). The AKTA Explorer plumbing system and both columns weresanitized with 1 M HCl and 1 M NaOH before use. The diluted reactionmixture was first loaded onto the Q-HP column that had been equilibratedwith 15 column volumes of 20 mM MES, pH 6.0. Unreacted OGF but notmono-[mPEG2-CAC-FMOC-40K]-[OGF] and unreacted PEG bound to the Q-HPresin and the conjugate and unreacted PEG were collected in the columnvoid fraction. Glacial acidic acid was added to the void fraction to afinal concentration of 5% (v/v) and the mixture was loaded onto theCG-71S column that had been equilibrated with 5% acetic acid/95% H₂O(v/v) (Solvent A). After sample loading, the column was washed with 10column volumes Solvent A to remove unreacted PEG. The conjugate waseluted with a linear gradient from 100% A to 20% A/80 B [Solvent B was5% acetic acid/95% acetonitrile (v/v)] over 10 column volumes with alinear flow rate of 90 cm/hour.

Fractions collected during reverse phase chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.09%TFA in water, and B, 0.04% TFA in acetonitrile. An Agilent PoroshellSB-300 C8 column (2.1 mm×75 mm) was used with a flow rate of 0.5 ml/minand a column temperature of 60° C. Detection was carried out at 280 nm.The column was equilibrated in 0% B and conjugate separation wasachieved using the gradient timetable shown in T OGF2.1.

Time (min) % Mobile phase A % Mobile phase B 0.00 100.0 0.0 3.00 100.00.0 5.00 80.0 20.0 16.00 40.0 60.0 19.00 40.0 60.0 25.00 20.0 80.0 28.0020.0 80.0

Fractions containing pure mono-[mPEG2-CAC-FMOC-40K]-[OGF] as determinedby analytical RP-HPLC were pooled, lyophilized and stored at −80° C.

A typical CG71S reversed phase chromatogram is shown in FIG. OGF2.1.RP-HPLC analysis of the purified conjugate is shown in FIG. OGF2.2, andMALDI-TOF analysis of the purified conjugate is shown in FIG. OGF2.3.The purity of the mono-PEG-conjugate was 100% by RP-HPLC analysis. Themass as determined by MALDI-TOF was within the expected range.FIG. OGF2.1. Typical CG71S reversed phase purification profile ofmono-[mPEG2-CAC-FMOC-40K]-[OGF]. The mono-PEGylated conjugate andunreacted PEG are indicated.FIG. OGF2.2. Purity analysis of [mono]-[CAC-PEG2-FOMC-40K]-[OGF] byreversed phase HPLC. The purity of the purified conjugate is determinedto be 100% at 280 nm.FIG. OGF2.3. MALDI-TOF spectrum of purifiedmono-[mPEG2-FMOC-CAC-40K]-[OGF]. The peak at 41997.4 Da is within theexpected range for the molecular weight of the mono-PEG-conjugate. Thevery weak signal is due to the absence of a positive charge on theconjugate.

E OGF3 PEGylation of Opioid Growth Factor (OGF) with[mPEG2-C2-FMOC-NHS-40K]

Stock solutions of 2.0 mg/mL OGF and 200 mG/mL mPEG2-C2-FMOC-NHS-40Kwere prepared in 2 mM HCl. To initiate a reaction, the two stocksolutions and a 0.5 M MES, pH 6.0, stock solution were brought to 25° C.and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.25 mg/mL OGF (2.2 mM), 20 mM MES and a1.25-fold molar excess of OGF over mPEG2-C2-FMOC-NHS-40K. After 3 hoursat 25° C. the reaction was quenched with 100 mM glycine in 100 mM HCl(10 mM final glycine concentration) for 10 minutes. The quenchedreaction mixture was diluted with deionized sterile H₂O until theconductivity of the diluted reaction mixture was below 0.5 mS/cm, andthe pH was then adjusted to 6.0 with 1 M NaHCO₃/Na₂CO₃, pH 10.0.

The mono-PEGylated conjugate was purified from the diluted reactionmixture by anion exchange chromatography using a column packed with Q-HPmedia (GE Healthcare) and reversed phase chromatography using a columnpacked with CG17S media (Rohm Haas) on an AKTA Explorer 100 system (GEHealthcare). The AKTA Explorer plumbing system and both columns weresanitized with 1 M HCl and 1 M NaOH before use. The diluted reactionmixture was first loaded onto the Q-HP column that had been equilibratedwith 15 column volumes of 20 mM MES, pH 6.0. Unreacted OGF but notmono-[mPEG2-C2-FMOC-40K]-[OGF] and unreacted PEG bound to the Q-HP resinand the conjugate and unreacted PEG were collected in the column voidfraction. Glacial acidic acid was added to the void fraction to a finalconcentration of 5% (v/v) and the mixture was loaded onto the CG-71Scolumn that had been equilibrated with 10 column volumes of 5% aceticacid/95% H₂O (v/v) (Solvent A). After sample loading, the column waswashed with 6 column volumes 5% acetic acid/20% ethanol/75% H₂O (v/v/v)to elute unreacted PEG. The conjugate was eluted with a linear gradientfrom 100% A to 100% B [Solvent B was 5% acetic acid/95% acetonitrile(v/v)] over 10 column volume with a linear flow rate of 90 cm/hour.

Fractions collected during reverse phase chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.09%TFA in water, and B, 0.04% TFA in acetonitrile. An Agilent PoroshellSB-300 C8 column (2.1 mm×75 mm) was used with a flow rate of 0.5 ml/minand a column temperature of 60° C. Detection was carried out at 280 nm.The column was equilibrated in 0% B and conjugate separation wasachieved using the gradient timetable shown in T OGF3.1.

Time (min) % Mobile phase A % Mobile phase B 0.00 100.0 0.0 3.00 100.00.0 5.00 80.0 20.0 16.00 40.0 60.0 19.00 40.0 60.0 25.00 20.0 80.0 28.0020.0 80.0

Fractions containing pure mono-[mPEG2-C2-FMOC-40K]-[OGF] as determinedby analytical RP-HPLC were pooled, lyophilized and stored at −80° C.

A typical CG71S reversed phase chromatogram is shown in FIG. OGF3.1.RP-HPLC analysis of the purified conjugate is shown in FIG. OGF3.2, andMALDI-TOF analysis of the purified conjugate is shown in FIG. OGF3.3.The purity of the mono-[mPEG2-C2-FMOC-40K]-[OGF] was 97.1% by RP-HPLCanalysis. The mass as determined by MALDI-TOF was within the expectedrange.FIG. OGF3.1. Typical CG71S reverse phase purification profile ofmono-[mPEG2-C2-FMOC-40K]-[OGF]. The mono-PEGylated conjugate andunreacted PEG are indicated. The resin was overloaded upon sampleloading and mono-[mPEG2-C2-FMOC-40K]-[OGF] was found in the voidfraction. The void fraction containing the conjugate was reloaded ontothe CG71S column and the conjugate was eluted in a second reversed phasechromatography run (data not shown).FIG. OGF3.2. Purity analysis of mono-[mPEG2-FMOC-C2-40K]-[OGF] byreversed phase HPLC. The purity of the purified conjugate is determinedto be 97.1% at 280 nm. The peak at 8.15 minutes is OGF.FIG. OGF3.3. MALDI-TOF spectrum of purifiedmono-[mPEG2-FMOC-C2-40K]-[OGF]. The peak at 41322.1Da is within theexpected range for the molecular weight of the mono-PEG-conjugate. Thevery weak signal is due to the absence of a positive charge on theconjugate.

E OGF4 PEGylation of Opioid Growth Factor (OGF) with[mPEG-Butyraldehyde-30K]

Stock solutions of 2.0 mg/mL OGF and 200 mG/mL mPEG-Butyraldehyde-30Kwere prepared in 2 mM HCl. To initiate a reaction, the two stocksolutions and a 1 M HEPES, pH 7.0, stock solution were brought to 25° C.and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.25 mg/mL OGF (2.2 mM), 20 mM HEPES and a1.25-fold molar excess of OGF over mPEG-Butyraldehyde-30K. After 15minute reaction at 25° C., a 50-fold molar excess of NaBH₃CN over PEGwas added, and the reaction was allowed to continue for an additional 16hours at 25° C. After 16 hr 15 min total reaction time, the reaction wasquenched with 100 mM glycine in 100 mM HCl (10 mM final glycineconcentration) for 10 minutes. The reaction mixture was diluted withdeionized sterile H₂O until the conductivity of the diluted reactionmixture was below 0.5 mS/cm, and the pH was then adjusted to 7.0 with 1M NaHCO₃/Na₂CO₃, pH 10.0.

The mono-PEGylated conjugate was purified from the diluted reactionmixture by anion exchange chromatography using a column packed with Q-HPmedia (GE Healthcare) and reversed phase chromatography using a columnpacked with CG17S media (Rohm Haas) on an AKTA Explorer 100 system (GEHealthcare). The AKTA Explorer plumbing system and both columns weresanitized with 1 M HCl and 1 M NaOH before use. The diluted reactionmixture was first loaded onto the Q-HP column that had been equilibratedwith 15 column volumes of 20 mM HEPES, pH 7.0. Unreacted OGF but notmono-[mPEG-Butyraldehyde-30K]-[OGF] and unreacted PEG bound to the Q-HPresin and the conjugate and unreacted PEG were collected in the columnvoid fraction. Glacial acidic acid was added to the void fraction to afinal concentration of 5% (v/v) and the mixture was loaded onto theCG-71S column that had been equilibrated with 5% acetic acid/95% H₂O(v/v) (Solvent A). After sample loading, the column was washed with 10column volumes Solvent A to remove unreacted PEG. The conjugate waseluted with a linear gradient from 100% A to 20% A/80% B [Solvent B was5% acetic acid/95% acetonitrile (v/v)] over 20 column volumes with alinear flow rate of 90 cm/hour.

Fractions collected during reverse phase chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.09%TFA in water, and B, 0.04% TFA in acetonitrile. An Agilent PoroshellSB-300 C8 column (2.1 mm×75 mm) was used with a flow rate of 0.5 ml/minand a column temperature of 60° C. Detection was carried out at 280 nm.The column was equilibrated in 0% B and conjugate separation wasachieved using the gradient timetable shown in T OGF4.1.

Time (min) % Mobile phase A % Mobile phase B 0.00 100.0 0.0 3.00 100.00.0 5.00 80.0 20.0 16.00 40.0 60.0 19.00 40.0 60.0 25.00 20.0 80.0 28.0020.0 80.0

Fractions containing pure mono-[mPEG-ButALD-30K]-[OGF] as determined byanalytical RP-HPLC were pooled, lyophilized and stored at −80° C.

A typical CG71S reversed phase chromatogram is shown in FIG. OGF4.1.RP-HPLC analysis of the purified conjugate is shown in FIG. OGF4.2. Thepurity of the mono-[mPEG-ButALD-FMOC-30K]-[OGF] was 95.3% by RP-HPLCanalysis.FIG. OGF4.1. Typical CG71S reversed phase purification profile ofmono-[mPEG-Butyraldehyde-30K]-[OGF]. The mono-PEGylated conjugate isindicated. The resin was overloaded upon sample loading andmono-[mPEG-Butyraldehyde-30K]-[OGF] was found in the void fraction. Thevoid fraction containing the conjugate was reloaded onto the CG71Scolumn and the conjugate was eluted in a second reversed phasechromatography run (data not shown).FIG. OGF4.2. Purity analysis of mono-[mPEG-ButyrAldehyde-30K]-[OGF] byreversed phase HPLC. The purity of the purified conjugate is determinedto be 95.3% at 280 nm. The peak with retention time at 1.69 minutes wasacetic acid derived from CG71S reversed phase chromatography.

E OGF5 PEGylation of Opioid Growth Factor (OGF) with [mPEG-Epoxide-5K]

Stock solutions of 2.0 mg/mL OGF and 200 mG/mL mPEG-epoxide-5K wereprepared in 2 mM HCl. To initiate a reaction, the two stock solutionsand a 0.5 M MES, pH 6.0, stock solution were brought to 25° C. and thethree stock solutions were mixed (PEG reagent added last) to give finalconcentrations of 1.25 mg/mL OGF (2.2 mM), 20 mM MES and a 1.25-foldmolar excess of OGF over mPEG-epoxide-5K over OGF. After 15 hours at 25°C. the reaction was quenched with 100 mM glycine in 100 mM HCl (10 mMfinal glycine concentration) for 10 minutes. The quenched reactionmixture was diluted with deionized sterile H₂O until the conductivity ofthe diluted reaction mixture was below 0.5 mS/cm, and the pH was thenadjusted to 6.0 with 1 M NaHCO₃/Na₂CO₃, pH 10.0.

The mono-PEGylated conjugate was purified from the diluted reactionmixture by anion exchange chromatography using a column packed with Q-HPmedia (GE Healthcare) and reversed phase chromatography using a columnpacked with CG17S media (Rohm Haas) on an AKTA Explorer 100 system (GEHealthcare). The AKTA Explorer plumbing system and both columns weresanitized with 1 M HCl and 1 M NaOH before use. The diluted reactionmixture was first loaded onto the Q-HP column that had been equilibratedwith 15 column volumes of 20 mM MES, pH 6.0. Unreacted OGF but notmono-[mPEG2-CAC-FMOC-40K]-[OGF] and unreacted PEG bound to the Q-HPresin and the conjugate and unreacted PEG were collected in the columnvoid fraction. Glacial acidic acid was added to the void fraction to afinal concentration of 5% (v/v) and the mixture was loaded onto theCG-71S column that had been equilibrated with 5% acetic acid/95% H₂O(v/v) (Solvent A). After sample loading, the column was washed with 10column volumes Solvent A to remove unreacted PEG. The conjugate waseluted with a linear gradient from 100% A to 20% A/80% B [Solvent B was5% acetic acid/95% acetonitrile (v/v)] over 10 column volumes with alinear flow rate of 90 cm/hour.

Fractions collected during reverse phase chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.09%TFA in water, and B, 0.04% TFA in acetonitrile. An Agilent PoroshellSB-300 C8 column (2.1 mm×75 mm) was used with a flow rate of 0.5 ml/minand a column temperature of 60° C. Detection was carried out at 280 nm.The column was equilibrated in 0% B and conjugate separation wasachieved using the gradient timetable shown in T OGF5.1.

Time (min) % Mobile phase A % Mobile phase B 0.00 100.0 0.0 3.00 100.00.0 5.00 80.0 20.0 16.00 40.0 60.0 19.00 40.0 60.0 25.00 20.0 80.0 28.0020.0 80.0

Fractions containing pure mono-[mPEG-epoxide-5K]-[OGF] as determined byanalytical RP-HPLC were pooled, lyophilized and stored at −80° C. Atypical GC71S reversed phase chromatogram is shown in FIG. OGF5.1.RP-HPLC analysis of the purified conjugate is shown in FIG. OGF5.2. Thepurity of the mono-[mPEG-epoxide-5K]-[OGF] was 100% by RP-HPLC analysis.

FIG. OGF5.1. Typical CG71S reversed phase purification profile ofmono-[mPEG-epoxide-5K]-[OGF]. The mono-PEGylated conjugate is indicated.FIG. OGF5.2. Purity analysis of mono-[mPEG-epoxide-5K]-[OGF] by reversedphase HPLC. The purity of the purified conjugate is determined to be100% at 280 nm.

E OGF6 PEGylation of Opioid Growth Factor (OGF) with[mPEG-Butyraldehyde-10K]

Stock solutions of 2.0 mg/mL OGF and 200 mG/mL mPEG-Butyraldehyde-10Kwere prepared in 2 mM HCl. To initiate a reaction, the two stocksolutions and a 1 M HEPES, pH 7.0, stock solution were brought to 25° C.and the three stock solutions were mixed (PEG reagent added last) togive final concentrations of 1.25 mg/mL OGF (2.2 mM), 20 mM HEPES and a1.25-fold molar excess of OGF over mPEG-Butyraldehyde-10K. After 15minute reaction at 25° C., a 50-fold molar excess of NaBH₃CN over PEGwas added, and the reaction was allowed to continue for an additional 6hours at 25° C. After 6 hr 15 min total reaction time, the reaction wasquenched with 100 mM glycine in 100 mM HCl (10 mM final glycineconcentration) for 10 minutes. The reaction mixture was diluted withdeionized sterile H₂O until the conductivity of the diluted reactionmixture was below 0.5 mS/cm, and the pH was then adjusted to 7.0 with 1M NaHCO₃/Na₂CO₃, pH 10.0.

The mono-PEGylated conjugate was purified from the diluted reactionmixture by anion exchange chromatography using a column packed with Q-HPmedia (GE Healthcare) and reversed phase chromatography using a columnpacked with CG17S media (Rohm Haas) on an AKTA Explorer 100 system (GEHealthcare). The AKTA Explorer plumbing system and both columns weresanitized with 1 M HCl and 1 M NaOH before use. The diluted reactionmixture was first loaded onto the Q-HP column that had been equilibratedwith 15 column volumes of 20 mM HEPES, pH 7.0. Unreacted OGF but notmono-[mPEG-Butyraldehyde-10K]-[OGF] and unreacted PEG bound to the Q-HPresin and the conjugate and unreacted PEG were collected in the columnvoid fraction. Glacial acidic acid was added to the void fraction to afinal concentration of 5% (v/v) and the mixture was loaded onto theCG-71S column that had been equilibrated with 5% acetic acid/95% H₂O(v/v) (Solvent A). After sample loading, the column was washed with 10column volumes Solvent A to remove unreacted PEG. The conjugate waseluted with a linear gradient from 100% A to 20% A/80% B [Solvent B was5% acetic acid/95% acetonitrile (v/v)] over 20 column volumes with alinear flow rate of 90 cm/hour.

Fractions collected during reversed phase chromatography were analyzedusing analytical reversed-phase HPLC. The mobile phases were: A, 0.09%TFA in water, and B, 0.04% TFA in acetonitrile. An Agilent PoroshellSB-300 C8 column (2.1 mm×75 mm) was used with a flow rate of 0.5 ml/minand a column temperature of 60° C. Detection was carried out at 280 nm.The column was equilibrated in 0% B and conjugate separation wasachieved using the gradient timetable shown.

Time (min) % Mobile phase A % Mobile phase B 0.00 100.0 0.0 3.00 100.00.0 5.00 80.0 20.0 16.00 40.0 60.0 19.00 40.0 60.0 25.00 20.0 80.0 28.0020.0 80.0

Fractions containing pure mono-[mPEG-ButALD-10K]-[OGF] as determined byanalytical RP-HPLC were pooled, lyophilized and stored at −80° C. Atypical CG71S reversed phase chromatogram is shown in FIG. OGF6.1.RP-HPLC analysis of the purified conjugate is shown in FIG. OGF6.2. Thepurity of the mono-[mPEG-ButALD-FMOC-10K]-[OGF] was 100% by RP-HPLCanalysis.

FIG. OGF6.1. Typical CG71S reversed phase purification profile ofmono-[mPEG-Butyraldehyde-10K]-[OGF]. The mono-PEGylated conjugate isindicated. The resin was overloaded upon sample loading andmono-[mPEG-Butyraldehyde-10K]-[OGF] was found in the void fraction. Thevoid fraction containing the conjugate was reloaded onto the CG71Scolumn and the conjugate was eluted in a second reversed phasechromatography run.

FIG. OGF6.2. Purity analysis of mono-[mPEG-ButyrAldehyde-10K]-[OGF] byreversed phase HPLC. The purity of the purified conjugate is determinedto be 100% at 280 nm. The peak with retention time at 1.7 minutes wasacetic acid derived from CG71S reversed phase chromatography.

E OGF7 Radioligand Competition Binding Assay for OGF Series at Mu andDelta Opioid Receptors

The binding affinities of OGF (control) and PEG-OGF releasableconjugates were evaluated using radioligand binding assays in membranesprepared from CHO-K1 cells expressing recombinant human μ or δ opioidreceptors.

Competition binding experiments were conducted by incubating membraneprotein to equilibrium in triplicate in the presence of a fixedconcentration of radioligand and increasing concentrations (0.01 nM to10 μM) of test compound in 100 μL final volume. The radioligands usedwere specific for each receptor type, and the assay conditions aredescribed in T OGF7.2. Following incubations, the membranes were rapidlyfiltered through GF/B filter plate (presoaked with 0.5%polyethyleneimine), washed four times with cold 50 mM Tris-HCl, pH 7.5,and the bound radioactivity was then measured. Non-specific binding wasmeasured in the presence of excess naloxone (100 μM); this value wassubtracted from the total binding to yield the specific binding at eachtest concentration.

For the releasable PEG-OGF conjugates, the receptor-binding activity ofboth released OGF and PEG-OGF (unreleased) conjugates was tested. Thetest compounds were stored under acidic condition to stabilize the PEGconjugation. To test the activity of PEG-OGF conjugates, the sample wasdiluted on the day of the assay. To test the activity of released OGF,two samples were prepared prior to the assay based on pre-determinedrelease rates (refer to T OGF7.3); one sample was diluted 10-fold inassay buffer (pre-incubated under physiological-like conditions for aperiod until ˜50% of OGF was estimated to be released) and the othersample was diluted 5-fold in 800 mM lysine solution, pH 10.0(pre-incubated under forced release conditions for less than 24 hoursuntil ˜95% of OGF was estimated to be released).

IC₅₀ (concentration of test compound required to inhibit 50% of specificbinding) values were obtained from non-linear regression analysis ofdose-response curves, using GraphPad's Prism 5.01 software, and werecalculated for those compounds that showed >50% inhibition of specificbinding at the highest concentration tested. K_(i) (affinity of testcompound) was obtained using the Cheng Prusoff correction usingexperimental K_(d) (affinity of radioligand) values that were previouslydetermined under these assay conditions.

The binding affinities of OGF and PEG-OGF conjugates are shown in TableOGF7.1. Opioid growth factor displayed similar, high affinity (1.3-2.0nM) for human μ and δ opioid receptors.

Since the releasable conjugates were pre-incubated, OGF was alsopre-incubated for the maximum period to test the activity of the peptideitself under the pre-incubation treatment conditions. As shown in FIG.OGF7.1, OGF remained stable following pre-incubation underphysiological-like (160 hours at 37° C., pH 7.5) and forced releaseconditions (16 hours at 37° C., pH 10.0). Pre-incubated OGF displayedsimilar, high affinity for μ and δ opioid receptors when compared to thecontrol prepared on the day of the assay (T OGF7.1).

Following pre-incubation of mono-mPEG2-CAC-40K-OGF for 160 hours andmono-mPEG2-C2-40K-OGF for 68 hours under physiological-like conditions,affinity for μ and δ opioid receptors was increased (compared to PEG-OGFconjugates prepared on the day of the assay) and regained (FIG. OGF7.2);OGF released from these conjugates retained receptor binding activity asshown by <9-fold loss in affinity relative to OGF. Similarly, bothPEG-OGF conjugates treated under forced release conditions displayedrelease of active OGF and high affinity binding to μ and δ opioidreceptors as shown by <4-fold loss in affinity relative to OGF.

The mono-mPEG2-CAC-40K-OGF conjugate displayed much lower affinity forboth receptors; reduction in affinity was 135 to 150-folds less relativeto OGF. The mono-mPEG2-C2-40K-OGF conjugate displayed a 2-fold reductionin affinity at the μ opioid and δ opioid receptor; this slight loss inaffinity suggests that the mono-mPEG2-C2-40K linker may have beenunstable and resulted in faster release of OGF under the assayconditions.

For the free PEGs (CAC-40K-fulvene and C2-40K-fulvene), affinity for μand δ opioid receptors was not seen as expected. As shown in FIG.OGF7.3, binding affinity could not be determined for the free PEGssince >50% inhibition of specific binding was not achieved up to thehighest test concentration (10 μM).

FIG. OGF7.1. Competition binding assay of OGF at human (A) μ opioid and(B) δ opioid receptors: effects of incubation treatment conditions. Datapresented as mean (±SEM) percent specific binding.FIG. OGF7.2. Competition binding assay of OGF and PEG-OGF conjugates(released and unreleased) at human (A) μ opioid and (B) δ opioidreceptors. Data presented as mean SEM) percent specific binding.FIG. OGF7.3. Competition binding assay of OGF and free PEGs at human (A)μ opioid and (B) δ opioid receptors. Data presented as mean (±SEM)percent specific binding.

TABLE OGF7.1 Summary of binding affinities for OGF, PEG-OGF conjugates,and free PEG. μ Opioid Receptor Fold δ Opioid Receptor Change FoldChange Relative Relative to Compound Ki (nM) to OGF Ki (nM) OGF OGF 1.51.0 1.8 1.0 OGF (Pre-incubated) 1.3 0.8 1.7 1.0 Mono-mPEG2-FMOC- 10.87.2 15.2 8.6 CAC-40K-OGF (Pre-incubated) Mono-mPEG2-FMOC- 4.3 2.9 3.52.0 C2-40K-OGF (Pre-incubated) CAC-40K-fulvene Not Not Not Not obtained(Free PEG) obtained obtained obtained C2-40K-fulvene Not Not Not Notobtained (Free PEG) obtained obtained obtained OGF (Forced release) 1.30.9 2.0 1.1 Mono-mPEG2-FMOC- 5.8 3.9 6.5 3.7 CAC-40K-OGF (Forcedrelease) Mono-mPEG2-FMOC- 3.3 2.2 3.2 1.8 C2-40K-OGF (Forced release)Mono-mPEG2-FMOC- 223.9 149.9 237.3 134.6 CAC-40K- OGF Mono-mPEG2-FMOC-3.2 2.2 2.6 1.5 C2-40K-OGFNot obtained=K_(i) values could not be determined since >50% inhibitionof specific binding was not achieved at the highest concentrationtested.

TABLE OGF7.2 Assay conditions. Non- Receptor Membrane specific Recepto

Source Protein Radioligand K_(d) binding Methods μ Human  5 μg/well [³H]2.0 nM Naloxone Reaction in 50 mM Tris- Opioid recombinant Naloxone (100μM) HCl (pH 7.5) at 25° C. for 1 h CHO-K1 (5 nM) on plate shaker cells δHuman 15 μg/well [³H] 3.0 nM Naloxone Reaction in 50 mM Tris- Opioidrecombinant DPDPE (100 μM) HCl (pH 7.5), 5 mM CHO-K1 (5 nM) MgCl₂, 0.1%BSA at 25° C. cells for 1 h on plate shaker

indicates data missing or illegible when filed

TABLE OGF7.3 Compounds. Stock conc. based on OGF Pre- Forced MW peptideStorage Release incubation release Compound (Da) (mg/mL) buffer ratecondition condition OGF 574 2.0 100 mM — 160 h in 50 mM 16 h in 800 mMHEPES Tris- lysine, pH HCl, 5 mM 10.0 at MgCl2, 37° C. 0.1% BSA, pH 7.5at 37° C. Mono-mPEG2- 41,332 4.4  2 mM 7.7% 160 h in 50 mM 16 h in 800mM FMOC-CAC- HCl after 68 h Tris- lysine, pH 40K-OGF; at 37° C. in HCl,5 mM 10.0 at releasable PEG 150 mM MgCl2, 37° C. Pi + 150 mM 0.1% BSA,NaCl, pH 7.5 at pH 7.4. 37° C. 95% within 24 h in 200 mM lysine, pH 10.0Mono-mPEG2- 41,332 5.0  2 mM 46% after 68 h in 50 mM 16 h in 800 mMFMOC-C2-40K- HCl 48 h at Tris- lysine, pH OGF; Releasable 37° C. in HCl,5 mM 10.0 at 150 mM MgCl2, 37° C. Pi + 150 mM 0.1% BSA, NaCl, pH 7.5 atpH 7.4. 37° C. 97.8% within 24 h in 200 mM lysine, pH 10.0FIG. OGF7.1. Competition binding assay of OGF at human (A) μ opioid and(B) δ opioid receptors: effects of incubation treatment conditions.FIG. OGF7.2. Competition binding assay of OGF and PEG-OGF conjugates(released and unreleased) at human (A) μ opioid and (B) δ opioidreceptors.FIG. 3. Competition binding assay of OGF and free PEGs at human (A) μopioid and (B) δ opioid receptors.

E INS1 Conjugation of Insulin with Dextran TetraethyleneGlycol-ButyrALD-40K

Insulin contains three primary amine groups, all of which can undergo areductive amination reaction with dextran tetraethyleneglycol-butyrALD-40K (dextran-butyrALD-40K). Reactions of insulin withdextran-butyrALD-40K therefore produce a mixture of mono-, di- andtri-conjugated peptides. The relative yields of the mono-, di- andtri-conjugated peptides depend primarily on the molar ratios of insulinand the dextran reagent used in the reactions and the reactionconditions (e.g., reaction time and temperature). The relative yield ofthe mono-conjugated peptide was determined to be very low unlessreaction conditions were selected in which the majority of the insulinremained unreacted. In order to increase the relative and absoluteyields of mono-conjugated insulin, a fraction of the amine groups on thepeptide were blocked by acetylation prior to reacting the peptide andthe dextran reagent. This example will describe the conjugation of bothpartially acetylated and non-acetylated insulin.

Conjugation of Partially Acetylated Insulin with Dextran-butyrALD-40K

Stock solutions of 2.5 mg/mL (430 μM) insulin, 2.24 mg/mL (8.62 mM)sulfo-N-hydroxysuccinimide (NHS)-acetate, and 138 mg/mL (3.45 mM)dextran-butyrALD-40K were prepared in DMSO/TEA (95%:5%, v/v), DMSO, andDMSO/TEA (99.35%:0.65%, v/v), respectively. To initiate an acetylationreaction of insulin, in which a fraction of the amine groups on thepeptide are acetylated, the insulin and sulfo-NHS-acetate stocksolutions were brought to ambient temperature and mixed at a 4:1 ratio(v/v). After 30 min acetylation reaction with stirring, conjugation ofthe peptide with dextran-butyrALD-40K was initiated by the drop-wiseaddition of an equal volume of dextran stock solution to the acetylationreaction mixture under vigorous stirring. Tween-20 was then added to afinal concentration of 0.05% (v/v) and the reaction mixture was broughtto 37° C. with stirring. 20 min after Tween-20 addition, 1 M sodiumcyanoborohydride was added to a final concentration of 17 mM and thereaction was allowed to proceed with continued stirring for anadditional 20 hours at 37° C.

Dextran-butyrALD-40K-insulin was purified from the reaction mixture byanion-exchange chromatography using Q Sepharose FF (GE Healthcare). Uponcompletion of the conjugation reaction, the reaction mixture was diluted1:3 with 20 mM HEPES (pH 7) and the mixture was loaded onto a columnpacked with Q Sepharose FF resin. Purification buffers were as follows:Buffer A: 20 mM HEPES (pH 7), and Buffer B: 20 mM HEPES, 1.0 M sodiumchloride (pH 7). The resin was washed with Buffer B and equilibratedwith Buffer A prior to sample loading. After loading, the resin waswashed with 10 column volumes Buffer A. Conjugated and nonconjugatedpeptides were eluted using a two-step gradient consisting of 0 to 25%Buffer B over 25 column volumes and 25% to 75% Buffer B over 5 columnvolumes at a flow rate of 90 cm/h (FIG. INS1.1).

FIG. INS1.1 Typical anion-exchange chromatography profile of theconjugation reaction mixture with partially acetylated insulin.Fractions containing less substituted conjugates are indicated in thegrey box.Fractions containing lower molecular weight, less substituted conjugateswere identified by SDS-PAGE (FIG. INS1.2).FIG. INS1.2 SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis offractions containing dextran-butyrALD-40K-insulin collected fromanion-exchange chromatography. The fractions represented in the laneswithin the box on the gel image correspond to the fractions in the greybox in FIG. INS1.1. Dextran perturbs the gel migration of thedextran-peptide conjugates and the conjugates' band locations are notindicative of the conjugates' sizes. The molecular weights of thestandards are indicated in kDa.

Fractions containing less substituted conjugates (denoted by the boxesin FIGS. 1 and 2) were pooled, diluted 10-fold with 20 mM HEPES, pH 7(Buffer A), and applied to a second column packed with Q Sepharose FFresin for sample concentration. The resin was washed with Buffer B andequilibrated with Buffer A prior to sample loading.Dextran-butyrALD-40K-insulin was eluted using a linear gradient of 0-75%Buffer B over 3 column volumes at a flow rate of 90 cm/h (FIG. INS1.3).

FIG. INS1.3 Concentration of purified dextran-butyrALD-40K-insulin byanion-exchange chromatography. Fractions containingdextran-butyrALD-40K-insulin are indicated by the grey box. The peakeluting at 2350-2400 mL contains residual nonconjugated insulin that wasco-purified with the conjugate from the first anion-exchangechromatography run.

Fractions containing concentrated dextran-butyrALD-40K-insulin (denotedby the grey box in FIG. INS1.3) were pooled and lyophilized. SDS-PAGEanalysis of the pooled fractions indicated the presence of a significantamount of nonconjugated insulin (FIG. INS1.4, Lane 1). The nonconjugatedinsulin can be removed by selective precipitation of the conjugate froma water/DMSO solution (50/50, v/v) through the addition of an organicsolvent (for example, acetonitrile). Dextran-butyrALD-40K-insulin isless soluble than nonconjugated insulin in organic solvents andprecipitates upon addition of an organic solvent.

Lyophilized dextran-butyrALD-40K-insulin was dissolved in water to apeptide concentration of 2 mg/mL. An equal volume of DMSO was added tothe solution and after thorough mixing acetonitrile was added drop-wiseuntil the composition of the mixture was 25% water, 25% DMSO, and 50%acetonitrile (v/v/v). Precipitated conjugated insulin was collected bycentrifugation and re-dissolved in water. The final concentration ofnonconjugated insulin in the re-dissolved product was reduced to lessthan 1% of the total peptide amount (FIG. 4, Lane 2).

FIG. INS1.4. SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis ofpurified dextran-butyrALD-40K-insulin. Lane 1:Dextran-butyrALD-40K-insulin purified and concentrated by anion-exchangechromatography. Lane 2: Purified and concentrateddextran-butyrALD-40K-insulin after precipitation with acetonitrile. Themolecular weights of the standards are indicated in kDa. There-dissolved conjugate was lyophilized and stored at −80° C.Conjugation of Non-Acetylated Insulin with Dextran-butyrALD-40K

Stock solutions of 2 mg/ml insulin and 42/mL dextran-butyrALD-40K wereprepared in DMSO/TEA (95%:5%, v/v). To initiate a reaction, both stocksolutions were brought to ambient temperature and then mixed in equalvolumes. After 5 min reaction with stirring at ambient temperature, 1 Msodium cyanoborohydride was added to a final concentration of 20 mM andthe reaction was allowed to proceed with continued stir for 22 hours atambient temperature.

Dextran-butyrALD-40K-insulin was purified from the reaction mixture byanion-exchange chromatography using Q Sepharose FF (GE Healthcare). Uponcompletion of the conjugation reaction, the reaction mixture was diluted15-fold with 20 mM HEPES (pH 7) and the mixture was loaded onto a columnpacked with Q Sepharose FF resin. Purification buffers were as follows:Buffer A: 20 mM HEPES (pH 7), and Buffer B: 20 mM HEPES, 1.0 M sodiumchloride (pH 7). The resin was washed with Buffer B and equilibratedwith Buffer A prior to sample loading. After loading, the resin waswashed with 5 column volumes Buffer A. Conjugated and nonconjugatedpeptides were eluted using a linear gradient of 0-100% Buffer B over 10column volumes at a flow rate of 150 cm/h (FIG. INS1.5).

FIG. INS1.5 Typical anion-exchange chromatography profile of theconjugation reaction mixture with non-acetylated insulin. The conjugatedand non-conjugated (free) peptides are indicated. The blue linerepresents absorbance at 280 nm.

Fractions containing dextran-butyraldehyde-40K-insulin were pooled,dialyzed against water, lyophilized and stored at −80° C. Removal ofnonconjugated insulin from the conjugate sample can be performed byselective conjugate precipitation with an organic solvent as describedin the previous section describing the conjugation of partiallyacetylated insulin with dextran-butyrALD-40K.

E INS2

Receptor binding: In vitro binding of the Insulin-dextran conjugate. Thein vitro affinity of the insulin-dextran conjugate for the insulinreceptor was evaluated using radioligand binding assays in CHO cellsthat stably express the recombinant human insulin receptor (CHO-hIR).The CHO-hIR cell line was previously generated and characterized.CHO-hIR cells were plated in 24 well plates and washed with assay buffercontaining 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄,9 mM Glucose, 10 mMHEPES, 0.5% BSA, pH 8.0. Competition binding assays were conducted byincubating CHO-hIR cells with increasing concentrations of insulin,dextran insulin and glycine dextran and a fixed concentration (100 μM)of ¹²⁵I-labelled recombinant human insulin for 4 hours at 4° C. Cellswere washed to remove unbound ligands, solubilized with 0.2 N NaOH andbound radioactivity was counted using a gamma counter. Non-specificbinding was measured in the presence of excess cold insulin andsubtraction of this value from the total binding yielded the specificbinding at each test compound concentration. IC₅₀ values were obtainedfrom non-linear regression analysis of specific binding versusconcentration curves.

Results: The results of the in vitro competition binding assay are shownin FIG. INS2.1. Insulin and the Dextran-TEG-butyrlaldehyde-40K acetylinsulin conjugate bound to the insulin receptor with IC₅₀ values of 4.3nM and 174.9 nM respectively. Dextran conjugation thus resulted in a40-fold reduction in the binding affinity of insulin. The dextran itselfdid not display specific binding to the insulin receptor atconcentrations up to 1 μM. The insulin-dextran conjugate was 98% pureand contained upto 2% of free and acetylated insulin. It is possiblethat the specific binding observed with the insulin-dextran conjugatecould be result of the free insulin in the sample.

E INS3 Effect of Dextran Conjugated Insulin on the Blood Glucose Levelsin the db/db Diabetic Mice

Dextran conjugated insulin 250 ug/mouse was administered by i.p.injection into diabetic mouse that had elevated blood glucose levels. Atdifferent time points after dosing blood glucose levels were measured.

PBS saline solution and Dextran equivalent dose were administrated asnegative controls. Insulin 50 ug/mouse was injected as positive control.Insulin 5 ug/mouse was also given to a group of db/db mice (to test ifthe 2% free insulin in the 250 ug Dextran-insulin prep; ˜5 ug; wouldhave any effect).

PBS and Dextran injections did not decrease db/db mice glucose levelsthroughout the whole study.

Dextran-Insulin injections dramatically decreased db/db mice glucoselevels by ˜40-60% at 1 hr and 2 hr after administrations. However thiseffect could be due to the free insulin that was in the conjugatepreparation. Dextran-Insulin group did show slightly prolonged effectcompared to 5 ug/mouse insulin injections. (t INS3.1 and FIG. INS3.1).

TABLE INS3.1 Glucose levels in db/db mice after compound administration.Blood glucose levels in mg/dL were expressed in Mean and SEM (standarderror). Dex-Ins Dextran PBS 250 ug Ins 50 ug Ins 5 ug 1.75 mg (N = 4) (N= 4) (N = 5) (N = 5) (N = 5) Time (hr) Mean SEM Mean SEM Mean SEM MeanSEM Mean SEM 0 486 51 495 32 445 29 510 37 485 31 1 528 54 263 39 210 16265 51 565 32 2 582 9 192 36 150 15 352 60 565 22 4 597 1 462 30 550 25587 9 562 27 8 577 14 494 10 558 23 538 30 540 36 24 560 24 517 24 54119 538 36 531 40 FIG. INS3.1. Glucose levels after compoundadministration (0-8 hr).

1. A conjugate comprising a residue of a therapeutic peptide moietycovalently attached, either directly or through a spacer moiety of oneor more atoms, to a water-soluble, non-peptidic polymer.
 2. A conjugateof claim 1, wherein the polymer is a linear polymer.
 3. A conjugate ofclaim 1, wherein the polymer is a branched polymer.
 4. The conjugate ofclaim 1, wherein the therapeutic peptide moiety is recombinantlyprepared.
 5. The conjugate of claim 1, wherein the therapeutic peptidemoiety is prepared by chemical synthesis.
 6. The conjugate of claim 1,wherein the polymer is selected from the group consisting ofpoly(alkylene oxide), polyvinyl pyrrolidone), poly(vinyl alcohol),polyoxazoline, and poly(acryloylmorpholine).
 7. The conjugate of claim6, wherein the polymer is a poly(alkylene oxide).
 8. The conjugate ofclaim 7, wherein the poly(alkylene oxide) is a poly(ethylene glycol). 9.The conjugate of claim 8, wherein the poly(ethylene glycol) isterminally capped with an end-capping moiety selected from the groupconsisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substitutedalkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substitutedaryloxy.
 10. The conjugate of claim 8, wherein the poly(ethylene glycol)has a weight-average molecular weight in a range of from about 500Daltons to about 100,000 Daltons.
 11. The conjugate of claim 10, whereinthe poly(ethylene glycol) has a weight-average molecular weight in arange of from about 2000 Daltons to about 50,000 Daltons.
 12. Theconjugate of claim 11, wherein the poly(ethylene glycol) has aweight-average molecular weight in a range of from about 5000 Daltons toabout 40,000 Daltons.
 13. The conjugate of claim 1, wherein thewater-soluble, non-peptidic polymer is conjugated at an amino-terminalamino acid of the therapeutic peptide moiety.
 14. The conjugate of claim1, wherein the water-soluble, non-peptidic polymer is conjugated at acarboxy-terminal amino acid of the therapeutic peptide moiety.
 15. Theconjugate of claim 1, wherein the water-soluble, non-peptidic polymer isconjugated at an internal cysteine amino acid of the therapeutic peptidemoiety.
 16. The conjugate of claim 1, wherein the water-soluble,non-peptidic polymer is conjugated at an epsilon amino group of aninternal lysine amino acid of the therapeutic peptide moiety. 17.-19.(canceled)
 20. The conjugate of claim 1, wherein the therapeutic peptideresidue is covalently attached through a spacer moiety of one or moreatoms.
 21. The conjugate of claim 20, wherein the spacer moiety includesan amine linkage.
 22. The conjugate of claim 20, wherein the spacermoiety includes an amide linkage.
 23. The conjugate of claim 20, whereinthe spacer moiety includes a disulfide linkage.
 24. The compound ofclaim 1, wherein the therapeutic peptide residue is covalently attachedvia a stable linkage.
 25. The compound of claim 1, wherein thetherapeutic peptide residue is covalently attached via a releasablelinkage.
 26. The compound according to claim 1, wherein thewater-soluble, non-peptidic polymer is dextran.
 27. The compoundaccording to claim 1, wherein the therapeutic peptide comprises of aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-469.28. A pharmaceutical composition comprising a conjugate of claim 1 and apharmaceutically acceptable excipient.
 29. A method for making aconjugate of claim 1 comprising contacting, under conjugationconditions, a therapeutic peptide moiety with a polymeric reagentbearing a functional group
 30. A method of treatment comprisingadministering a compound of claim 1 to a subject in need thereof.